SF 253 
.S8 
Copy 1 



^HE LECITHIN CONTENT OF BUTTER 

AND ITS POSSIBLE RELATIONSHIP 

TO THE FISHY FLAVOR 



A THESIS 

Presented to the Faculty of the Graduate School 

OF Cornell University for the Degree of 

DOCTOR OF PHILOSOPHY 



BY 

GEORGE CORNELL SUPPLEE 



[Reprint from the Cornell University Experiment Station Memoir 29, November, 1919] 



THE LECITHIN CONTENT OF BUTTER 

AND ITS POSSIBLE RELATIONSHIP 

TO THE FISHY FLAVOR 



A THESIS 

Presented to the Faculty of the Graduate School 

OF Cornell University for the Degree of 

DOCTOR OF PHILOSOPHY 



BY 

GEORGE CORNELL SUPPLEE 



[Reprint from the Cornell University Experiment Station Memoir 29, November, 19 19] 



O' 



^>3 



X Of -.'. 



CONTENTS 

PAGE 

Previous inv<>stisafi()iis 101 

Lecithin cleconii)osition in butter as a possible eaus(^ of the fishy flavor lo4 

Chemical constitution, properties, and distribution of lecithin. . . . 104 

Theoretical discussion 106 

Investigational work 108 

Quahtative determination of lecitliin in butter 108 

Amount of lecithin in butter 110 

Trimethylamine salts of the fatty acids Ill 

Effect of working trimethylamine salts of the fatty acids into 

butter. ..........'.. ' 113 

Quantitative estimation of trimethylamine^ in fishy butter 123 

Development of fishy flavor in experimental butters 12(3 

Variation in acid value of experimental butters 129 

Trimethylamine and ammonia content of experimental butters. , 134 

Bacteriological studies 136 

Bacterial analysis 137 

Inoculations for the purpose of developing the fishy flavor. . 137 

Longevity of Bactermni ichthyosmius in butter 141 

Further studies with Bacterium ichthyosmius 142 

Trimethjdamine and ammonia production by Bacterium ich- 
thyosmius in milk and cream 144 

Production of trimethylamine from lecithin and choline^ by 

bacterial action 145 

Bacterial inoculations into l;)utterfat 146 

Summary 148 

Acknowledgments 149 

Bibliography 150 



97 



THE LECITHIN CONTENT OF BUTTER 
AND ITS POSSIBLE RELATIONSHIP TO THE FISHY FLAVOR 



THE LECITHIN CONTENT OF BUTTER 
AND ITS POSSIBLE RELATIONSHIP TO THE FISHY FLAVORS 

George Cornell Supplee 

The commercial value of butter is based to a great extent on its quality, 
which in turn is determined" by its flavor. The commercial grading of 
butter on this basis, and the recognition of certain characteristic defects, 
have resulted in the establishment of certain terms more or less descriptive 
of the flavors found. Among the terms commonh^ applied to the flavors 
in butter are such words as rnetaUic, fishy, oily, raucif}, (alloivj/. Since the 
presence of any of these flavors carries with it a reduction in commercial 
value of the butter, considerable effort has bc^en made to detei'mine their 
causes and prevent their development. Unfortunately most of these efforts 
have not met with a high degree of success. This may be ascribed to several 
reasons, among which are the following: lack of positive idcuitificatioTi of 
the same flavor by different investigators; lack of ad(>quate chemical 
methods for the isolation and measurement of the small amount of sub- 
stance capable of producing the flavor; and lack of coopei'ation between 
the chemist, the l^acteriologist, and the experienced butter judge. 

Fishiness in buttcM-, with which this investigation is primarily con- 
cerned, is usually (h^scribed as a flavor resembling that of salmon or 
mackerel, altho the names of other varieties of fish are occasionally used 
to describe the flavor more explicitly. While the typical fishy flavor in 
butter is readily recognized by experts, it is oftcni accompanied by a more 
or less oity condition whi(;h tends to create differen(;es of opinion as to its 
exact nature. But if the opinion of butter judges of long experience is to 
be considered as trustworthy, it may be said that the true fishy flavor is 
entirely distinct from the oily flavor even tho the oily condition may 
precede or accompany it. 

PREVIOUS INVESTIGATIONS 

The earher investigations bearing on fishiness in dairy products have 
been largely confined to milk and butter. One instance is recordcKl, how- 
ever, in which this condition was observed and studied in evaporated milk. 

' Also presented to the Faculty of the Graduate School of Cornell University, December, 191 S, as a 
major thesis in partial fulfiUnient of the reiiuirenients for the degree of doctor of philosophy. 

101 



102 George Cornell Supplee 

Harding, Rogers, and Smith (1900)- report the fishy flavor in a sample 
of milk brought to the New York State Agricultural Experiment Station 
at Geneva in 1900. The source was traced to a single animal in the herd, 
but investigation failed to locate any pathological condition or any 
irregularity in the feeding which might cause the trouble. Attempts to 
reproduce the flavor by inoculating milk with bacteria isolated from this 
cow's udder also fail(Ml. The same authors mention also an instance 
brought to their attention by W. E. Griffith, in which a peculiar flavor 
developed in June butter after storage at 18° to 22° F. This flavor was 
described by butter experts as fishy. 

Piffard (1901) discusses the fishy flavor in dairy products, and suggests 
the possible relationship between certain algae found in stagnant water, 
and fishiness in milk and butter. His theory is supported l)y the fact 
that the flavor is often produced in water by the growth of algae and 
diatoms, and he lielieves, therefore, that cows having access to such 
water may transmit the condition to milk. Refening to tlie flavor in 
butter, he considers the idea that salt may be resi)onsil)le and mentions 
the ability of salt to absorb flavors and odors of materials stored near it. 

Harrison (1902), discussing butter defects at about the same time, 
states that the characteristic off-flavors of butter — fishy butter being 
specifically mentioned — are caused l)y the growth of undesirable bacteria 
in the cream. 

O'Callaghan (1902) published certain observations on fishiness in 
Australian butter. He states that he has found this condition in butter 
only two hours old. From his investigations he concludes that O'idium 
lad is is the causal agent. Later (O'Callaghan, 1908) he elal)orated on 
his former views, concluding that Oidium lactis associated with the lactic- 
acid bacteria in cream will usually produce a fishy flavor in the butter. 
He mentions the presence of the defect in uiLsalted butter, and recom- 
mends the improvement of sanitary conditions in the creameries, and 
pasteurization, as a remed}'. His conclusions have not been confirmed 
by other investigators. 

Rogers (1909), after a rather exhaustive stutiy of tiie occurrence and 
cause of fishy butter, confirms many observations commonly noted in 
connection with this ti'ouble but is unable to confirm the ol)servations 
of O'Callagiian. He also s-emingly eliminates the theory that trimethyl- 

- Dates in parenthesis refer to Biblioyraphy, page 150. 



The Lecithin Content of Butter 103 

amine is directly responsible for the flavor. After studying the effect of 
high-acid cream, overworking, and the consequent increased oxygen con- 
tent of the butter, and by conducting bacteriological investigations, he 
concludes that high-acid cream is essential to loring about the condition, 
altho he points out that not all l^utter made from such cream develops 
the fishy flavor. In this respect he states (page 20 of reference cited) 
that '^ fishy flavor may be produced with reasonable certainty by over- 
working the butter made from sour cream." From his viewpoint the 
probability that microorganisms are the cause falls into disfavor by the 
advancement of the opinion that " fishy flavor is caused by a slow, spon- 
taneous, chemical change to which acid is essential and which is favored 
by the presence of small amounts of oxygen " (page 20 of reference). 
More recently the same author (Rogei'S, 1914, a and b) points out that 
fishiness in butter may bo preceded by an oily or a metalhc flavor, and 
reiterates his views that the evidence is against the theory that the fishy 
condition is of a bacterial nature. He also states (1914b) that "fishy 
flavor is said to occur rarely or not at all in unsalted butter and it is 
possible that the salt furnishes certain conditions which are essential to 
the development of the fiavoi'." 

Reakes, Cuddie, anil Reid (1912) find no significant differences in the 
bacterial flora of fishy and of high-grade butter, and, in agreement with 
Rogers, state that " the development of fishy flavour in butter arises as 
a result of a chemical change inducing a splitting-up of some of the con- 
stituents into compovmds possessing this peculiar character of smell and 
taste, the factors responsil>le for such change being apparently a degree 
of high acidity of the cream and overworking." 

Hunziker (1916) states that high pasteurization temperatures (185° F.) 
when used on sour cream tend to produce a very i)Oor quality of butter, 
which often has a disagreeable oily taste suggestive of fishiness. He 
points out that this is particularly true when cows are on green pasture 
and the butterfat contains a rather high percentages of olein, which may 
b.' oxidized with relative ease in the presence of high temperatures and 
high acid. 

Hammer (1917) reports that he found a can of evaporated milk which 
possessed a marked fishy flavor and odor and fi-om which he was able to 
isolate an organism heretofoi-(? utulescribed. H(^ gives to this organism 
the name Bacterium ichthyosiniu-'^, which was suggested by Di-. A. W. 



104 George Cornell Supplee 

Dox. The flescription of the ori>;anisin seems to indicate that it is closely 
allied to the Proteus group. By inoculation experiments Hammer was 
able to reproduce the flavor in milk and cream under both aerobic and 
anaerobic conditions. He noted that the intensity of the odor was in- 
creased by the addition of alkali to the milk after the incubation period. 
He was unable, however, to produce fishiness in butter by direct inocula- 
tion or by inoculating the cream before churning. Bacteria counts at 
various intervals during the storage period showed an immediate decrease 
in numbers in salted butter, and an increase tluring the first few days in 
unsalted buttei- followed by a pronounced decreas(\ 

Washburn and Dahlberg (1918), while studying the influence of salt 
on storage butter, found that salted butter was more likely to turn fishy 
in stoi-age than was vmsalted butter, and furthermore that there appeared 
to be a tendency toward a progressive development of the flavor thru 
metallic to oily and finally to fishy. 

LECITHIN DECOMPOSITION IN BUTTER AS A POSSIBLE CAUSE OF THE 

FISHY FLAVOR 

CHEMICAL CONSTITUTION, PROPERTIES, AND DISTRIBUTION OF LECITHIN 

Lecithin, which stands in close relation to the fats, belongs to a more 
or less definite group of substances known as phosphatidt^s, or phos- 
phorized fats. These bodies appear to be a group of esters containing 
nitrogen, phosphoric acid, and fatty-acid radicals. Lecithin, wliich is 
the best known of the phosphatides, contains two fatty-acid radicals 
and the nitrogenous base choline, combined with glyccn-ophosphoric acid. 
According to the kintl of fatty acid present in the molecule, it is possible 
to have various types of lecithin, such as stearyl, palmityl, and oleyl. 
A number of investigators seem to agree that every true lecithin con- 
tains at least one oleic- acid radical. There seems to be uncertainty as 
to whether choline is th(> only base present in lecithin. MacLean (1909) 
was able to get only 42 per cent of the theoretical amount from lecithin 
isolated from heart muscle, and 65 per cent from k^cithin of egg yolk. 
Other investigators have found the same to hold true of lecithin from 
different sources. 

Lecithin has certain properties in common witli the fats, particularly 
with respect to its solvents. It diliers, however, by being less soluble 



The Lecithin Content of Butter 105 

in ether and more soluble in alcohol. It is precipitated from alcoholic 
solution by acetone; in water it swells to a colloidal mass which on micro- 
scopic examination appears as oily drops and threads. It saponifies 
with alkahes and baryta water, yielding the corresponding soaps, salts of 
glycerophosphoric acid, and choline. Hammarsten and Hedin (1915) 
state that it is slowly decomposed by dilute acids and enzymes (lipase). 
Barger (1914) states that Bacterium prodigiosus produces trimethylamine 
from chohne and lecithin; he also cites references to show that lecithin 
is decomposed during putrefaction, yielding fatty acids, glycerophosphoric 
acid, choline, and ultimately trimethylamine. Hasebroek (1888) claims 
that methylamine, ammonia, methane, and carbon dioxide may be finally 
produced from choline during putrefaction. On being heated with strong 
caustic soda or potash, lecithin yields trimethylamine, which has a dis- 
tinct fishy odor, this being one of the characteristic qualitative tests 
for it. Leathes (1913), in citing the work of various investigators, seems 
to think that lecithin is rather unstable. He thinks this property is 
due to the unsaturated oleic-acid radical contained, and offers this as 
the reason why the substance gives Pettenkofer's reaction. The work of 
Long (1908), however, seems to indicate that lecithin is more stable than 
has been generally believed. Koch (1902-03) has shown that various 
salts will cause lecithin to precipitate as a gelatinous mass, and that 
acids, if sufficiently dissociated (0.005 M sulfuric), will accomphsh the 
same thing. 

Lecithin seems to be widely found in nature, being present in many 
plant cells and animal fluids. It is particularly abundant in the brain, 
the nerve tissue, and the yolk of egg. It is also reported as existing in 
blood corpuscles, blood plasma, lymph, milk, and bile. Since the methods 
used for the quantitative^ estimation of lecithin depend on the deter- 
mination of phosphorus in alcoholic or alcohol and ether extracts, it is 
doubtful whether the figures given are absolutely correct due to the 
fact that other phosphatides are extracted and also because the empirical 
fornuila used in the calculation may be inaccurate for the particular 
lecithin Involved. Altho there have been conflicting statements as to 
whether milk contains lecithin, there seems to be sufficient evidence that 
it does. The results obtained by Nerking and Haensel (1908) are sub- 
mitted in table 1: 



106 



George Cornell Supplee 



TABLE 1. The Lecithin Content of Various ^NIilks 
(From Nerking and Haensel) 



Kind of milk 



Human, 10 samples 
Cow's, 17 samples. . 
Ass's, 6 samples. . . 
Ewe's, 4 samples . . 
Goat's, 11 samples. 
Mare's, 8 samples . 



Percentage of lecithin 



Highest 


Lowest 


Average 


080 


024 


0.050 


110 


036 


003 


o:]9 


0.000 


016 


107 


051 


083 


075 


030 


040 


017 


007 


Oil 



Glikin (1909), studyino; the lecithin mid iron content of milk, reports 
0.0515 per cent lecithin in whole milk, 0.05 per cent in cream, and 0.1329 
per cent in Iminaii milk. Fetzer (1911), studying the lecithin content 
of milk imder pathological conditions, finds (hat it is lower in milk from 
cows suffering with mastitis than in milk from normal cows. He finds 
also that the lecithin content decreases as the fat decreases. The work 
of Bordas and De Haczkowski (1902) indicates that the amount of lecithin 
varies witli the lactation ])eriod. They find that it is at the maximum 
at the bi'gimiing, and gradually decreases during the remainder of the 
period. Their observations were from seven cows. 

THEOKETieAL OISCI'SSIOX 

Trimethylaniine as a decomiiosition product of lecithin was brought 
to the attention of the writei- as a possible cause of the fishy flavor in 
butter about thrcH' y(\us ;igo, when he was working with lecithin isolated 
from brain tissue. Altlio tlicre seem to be no pul)lished data concerning 
trimethylaniine in relation to this subject, and Hogers (1909) claims 
that it can be work(Ml into l)utter in large amounts without producing 
the fishy tiavor, it is neveithdi'ss believed by many that this substance 
is in some way resp()nsil>le. On boiling l(>citliin isolated from brain 
tissue and egg yolk with stiong caustic soda. tli(> writ(a' has been able 
to obtain a distinct ()il\- ;ind fishy odor which \vas assei'ted by many to 
be typical of the odor of fishy buttei'. The only possible sourc(> of such 
an odor in this case w;!S tlu* t riniet hylnniine derived from the lecithin. 
This i-esult, together with the fact that there s(>ems to I;)e good evidence 
that lecithin is [iresent in milk, led to the assumption that the substance 



The Lecithin Content of Butter 107 

may exist also in butter and that by its decomposition it can slowly 
liberate trimethylamine with the consequent production of the fishy 
flavor and odor. 

Provided that lecithin can be shown to exist in butter, the above assump- 
tion is supported by several facts. It is well known that enzymes are 
capable of bringing about many chemical decompositions which result 
from the action of acids and alkalies on organic substances. It also appears 
to be a fact that butter may contain enzymes derived from the udder, 
and from the action of bacteria in the milk or the cream before it is made 
into butter, and furthermore that the activity of such enzymes is not 
entirely stopped at the temperature at which butter is stored. Hammar- 
sten and Hedin (1915) state that lecithin is decomposed by dilute acids 
and enzymes. The citations of Hasebroek (1888), Barger (1914), and 
others show that lecithin is decomposed by bacteria yielding choline, 
which finally yields trimethylamine. It is also a well-known fact that 
this substance in very small amounts possesses a distinct fishy odor, but 
in concentrated solution it has a strong ainmoniacal odor. Speaking of 
the former property, Davis (1912), quoting Tayloi', states that the " odour 
[referring to the peculiar fishy odor suggestive of herring brine] is grad- 
ually developed by adding lime to a solution of the base, but requires 
some time to reach its maximum intensity." 

In addition to the forc^going facts the writer has observed certain fea- 
tures that may have some beaiing on this problem. In inspecting butter 
u.sed in the Navy, it has been noticed that certain samples of cream evolved 
a peculiar fishy odor on the addition of alkali used for titration. This 
phenomenon was first brought to the attention of the writer by A. M. 
Besemer, and has since been confirmed by a number of men, some of 
whom have wide reputation as butter judges. Since trimethylamine is 
a base which is liberated from its acid combinations by alkalies, it is quite 
possible that the odor mentioned above was due to this substance's having 
been liberated from its acid combination in the cream. If such were 
the case, it is conceivable that butter made from such cream might, 
during storage, give up its trimethylamine thru the action of enzymes. 
In this connection it has been noted that certain samples of old butter, 
which wei-e not scored as fishy, when brought into contact with a warm 
solution of soap powder would give off a strong herring-like odor. This 
phenomenon might also be explained as in the case of the cream. In 
addition to these features it has been noted that certain samples of fishy 



108 George Cornell Supplee 

biittcn- may lose tluMr charaetoi-istic flavor aftcn- a period of time. This 
has also been observed by other investigatois. It is possible that this 
characteristic may be explained by the fact that, since trimethylamine 
is extremely volatile, it may pass off, or that the instability of the acid 
combination clianj^cs so that tlie conditions are not right for its manifes- 
tation. The writer has noted a very strong fishy odor in partially decom- 
pos(Ml egg yolk held at refrigerator temperature, which had entirely dis- 
appeared two wcH'ks later. 

On the basis of the foregoing facts and observations and the evident 
lack of contradiction of most of them with what is known about fishy 
butter, the following experimental woi'k was plamicd with the object 
of determining the possible i-elationship of trimethylamine to this flavor. 
In calling attention to the lecithin, it may be stated that the writer is 
cognizant of the fact that trimethylamine may Iw produced from other 
sui)stances. This material has been chosen as the object of study pri- 
marily Ix'cause there is exact knowledge concerning its cleavage and some 
of the agencies bringing this condition about. 

TXVESTIGATIONAL WORK 
QUALITATIVE DETERMINATION OF LECITHIN IN BUTTER 

The first experimental work undertaken was to demonstrate the pres- 
ence of lecithin in butter, since there ap[)ear(>d to be no rei)orts on this 
point in the hterature. One hundred gi'ains of melted butter was thoroly 
mixed with sufficient anhydrous calcium sulfate (alwut one kilogram) 
so that tlu^ mixtui'e retaininl its dry powdered form to sucli an extent 
that it could be icadily sifted l3etwe(ni the thumb and tlie finger. The 
mixture was transfencd to a sp(>ciallv constructed pcM'colator and extracted 
for 48 hours with D.Vpei-cent alcoh.ol at (50° (\ The alcoholic extract 
w.as evaporat(>d down and the residue was treated with a small amount 
of ether, wiiich rook uj) the fat, the fatty acids, ;uid part of the lecithin. 
The ])ait iiisohible in eth(M- was again taken up with warm alcohol, and 
what may i)e termed the lecithin Jradion was precipitatcnl by thoroly 
cooling \\\v alcoholic sohition. The substance thus obtained precipitated 
iTi the foiin of small, wart-like masses, which clung tenaciously to the 
sides of the })eakei'. On this material, which presumably contained a 
high proporiioii of lecithin, various observations were madf^ and qualita- 
tive tests applied. The following charactei'istics were noted: 



The Lecithin Content of Butter 109 

On drying at oi-dinary temperature and pressun^ tlie material appeared 
as a semi-amorphous and oily substance of a pale, dirty yellow color. 
It was entirely soluble in alcoliol but was partially thi'own out of solu- 
tion by the addition of an excess of ether. The precipitate formed in 
this manner was finely granular and was white in color. In water it 
formed a semi-colloidal solution which on microscopic examination ap- 
peared as minute oily droi)s. When the watery suspension was heated, 
the particles would cohere to form a sticky mass which changed to a 
distinct brown color. Both the dry substance and the watery suspension, 
when heated with strong caustic soda, gave off a marked fishy odor 
resembhng sometimes dried herring and sometimes salmon oil. This 
observation was in the great majority of cases confirmed by a number 
of colleagues. The fishy odor obtained from the material in this manner 
seemed to furnish positive evidence that lecithin was present. To further 
strengthen this behef, Pettenkofer's test with sugar and sulfuric acid was 
applied to the dry material with positive results. The above observations 
were confirmed with lecithin extracted from fresh butter, salted and 
unsalted, and from other miscellaneous samples of normal butter. 

Altho the evidence that lecithin exists in butter in del (K't able quanti- 
ties seemed conclusive, it was decided to determine, if }")ossible, the pres- 
ence of choline, which, as already pointed out, is one of the components 
of the lecithin molecule. This was accomplished by boiling the residue 
of the first alcohohc extract witli baryta water, which removed all fat, 
fatty acids, and fatty-acid radicals of the lecithin in the form of barimn 
soaps. After the barium soaps were filtered off, the excess barium was 
removed with carbon dioxide, the barium carbonate filtered off, and the 
filtrate containing choline^ and barium glycerophosphate evaporated to 
a sirupy consistency. This I'esidue was then treated witli absolute alcohol, 
in which choline is solubl(> but baiium glyeerophosj)hate is insoluble. 
On evaporation of the al)solute alcohol a small amount of sirupy sub- 
stance remained. To tliis material qualitative tests for choline were 
applied. The most characteristic of such tests is the periodide test 
described by Stanek (1905), which is made by adding a small amount 
of strong iodine solution (153 grams of iodine and 100 grams of potassium 
iodid(> in 200 grams of water) to an aqueous solution of choline. A posi- 
tive test is indicated by the formation of a brown precipitate of choline 
periodide, which on microscopic examination in the presence of the reagent 
appears as dark brown refractive and notched prisms or rhomboidal 



no 



George Cornell Supplee 



leaflets. On evaporation of the reap;ent the crystals lose their shape 
and appear to liquefy, forming l^rown, oily droi)lets which again assume 
their crystalline structure on the addition of more reagent. On the 
application of this test to the choline ol)tained from butter lecithin. ^^ 
was foimd that the results conformed in all respects to the descriptions 
of this periodide. The accompanying platc^ of photomicrogi'aphs (Plate 
VI) shows the characteristic crystals and oily droplets of the periodide 
foi-med by the cholin(> from flutter. In addition to this test it was shown 
that the small amount of eiioline obtained would give off a slight but 
distinct fisliy odor on being heated with solid caustic soda. 

AMOUNT OF lecithin IN BUTTKR 

Since the qualitative tests sc(Mned to leave no room for doubt as to 
the pr(\sence of lecithin in butter, the next step was the quantitative 
estimation of this sul)stance. In view of the evident (Hfhculty in securing 
an absolutely pure lecithin free from oth(>r phosphatides, the estimations 
were basc^d on the phosphorus content of extracts and the amount of 
lecithin calculated acconUng to thc^ formula of the distearyl type. The 
results of such determinations on various types of butter made from 
different lots of cream are shown in table 2: 

TABLIC 2. Lecithin Content in Vaiiious Butters* 



Sample 


Type of creani from 
which butter was made 


Age of 
butter 

(days) 


P^O, 
(per cent) 


Lecithin, 
distearyl 

type 
(per cent) 


1 


Raw sweet 

Pasteurized sweet 

Raw ripened 

Pasteurized ripened 

Raw sweet 

Pasteurized sweet 

Raw ripened 

Pasteurized ripened 

Raw sweet 


G 
(i 
(> 
G 

48 
48 
4S 
48 

72 
72 

72 
72 


0127 
0127 
0122 
007.5 

00i)2 
0120 
008(1 
{){)K', 

0111 
OOS'.t 
00S3 
0.009.5 


0.0723 


2 


0723 


3 


0G93 


4 


0133 


5 


()r.22 


6 


0G82 


7 


(M8S 


8 


0171 


9 


Otiol 


10 


Pasteurized sweet 

Raw ripened 


OoO.") 


11 


0471 


12 


Pasteurized ripened 


OMO 



* Thcsp figures were furnished by J. T. Cusick, fbeiiiist for the State Department of Agriculture, located 
at Cornell University. 



Memoir 29 



Plate VI 















°>^ \> 




^h 



^ \ 



I 



'. ,./ 



* \\ 



/ 



.^ 



-^^ 



^ 



CHOLINE PERIODIDE CiiYSTALS OBTAINED FROM CHOLINE OF BUTTER LECITHIN 
Photouiicro-^raplis, X 3S0 



The Lecithin Content of Butter 111 

A study of table 2 shows a fairly constant lecithin content in butter 
from various lots of cream and in different types of butter made from the 
same lot of cream. There is one feature, h()\v(>\-ei-, wliicli is worthy of 
note, and this is that in most instances there is a tendency toward a lower 
lecithin content in the ripen(>d-cream butter than in that made from 
unripened cream. This may be sio;nificant in the litiht of the statement 
by Hammarsten and Hedin (11)1")), that lecithin is decomposed by dilute 
acids and enzymes. This fact applied to these nsults mioht indicate 
that the acidity of the cream slowly decomposed the lecithin, and its 
decomposition products, particularly the tilycerophosphoric acid, were 
washed out with the buttermilk. If such wei-e the case it would be very 
easy to acc-ount for the lower phosphoi-us content in sour-cream butter. 

TRIMKTHVLAMIXK SALTS OF THE FATTY ACIDS 

It was decided that l^efore an attempt was made to correlate trimethyl- 
amine with the hshy flavor of butter, this substance should be prepared 
in a pure state and those characteristics determined which mifi;ht have 
a bearing on this joarticular jiroblem. Trimethylamine was made by 
heating 50 grams of ammonium chloi'ide and 440 grams of a 40-per-cent 
solution of formaldehyde in the autochive at 122° ('. for thirty minutes. 
Any excess formaldehyde was then exiK>ll(>d and th(> trimc^thylamine 
Uberated from its hydrochloride by distilling from an alkaline solution. 
A 10-per-cent solution was easily obtained at the oixlinary temperature 
and pressure. The trimethylamine thus procured was combined with 
lactic, butyiic. oleic, and stearic acids, and also with the mixed soluble 
and insoluble fatty acids obtaincMJ from butter according to the procedure 
outlined by Browne (1899). While \hv piopinties of these acid addition 
products seemed to offer an interesting fi<^ld for study, only such of their 
characteristics luv recorded here as might have a direct relationship to 
the fishy flavor in butter, namc^ly, their stability, volatility, and behavior 
in the pres(>nce of sodium chloride. 

The lactic-acid combination with (limethylamine proved to be a rela- 
tively stable oily licjuid })()ssessing no characteiistic taste other than 
that shown by many conunon salts. The odor, c>s()(H'ially after the liquid 
had been standing in a stoppei-ed bottle, seemed to be slightly i\shy. 
Evidence on this point is not conclusive because it is possible^ that this 
was due to excess trimethylamine added at the time of neutralization and 



112 George Cornell Supplee 

not shown by the indicator used. Tlc^atinji; socnu^d to intensify the odor 
to some extent, wliich would incUcute instabihty at high temperatures. 

The butyiic-acid (■oml)ination witli trimethylamine was a substance 
extremely volatile^ at ortUnar}' temperatures. Tlu^ odor greatly resembled 
fish oil; the taste resembling this product was manifest onh^ when very 
small amounts were us(>d, and then not regularly. 

Oleic acid and Irinu'thylamine formed a soft soai) whi(;h was very 
unstable as evidenced by the lilx'i'ation of the trimethylamine; the greater 
the dilution, however, th(^ less this condition was manifested. This 
soap could not \)c obtained entirely free from water, even at the ordinary 
temperatures, because of the sinuiltaneous gi\-ing-off of ti'imethylamine 
and watcn'. Tliis liberation was sut'li that nothing remained but the free 
acid. Fui'thermoi-e, on the addition of sodium chloi'ide to its water 
solution, the sodium ion i'eadil\" r(>i)iaced the trimethylamine radical, with 
the consequent jirecipitation of the sodium soap and the formation of 
trimethjdamine hydrochloride. 

The trimethylamine stt>arate showed the same charactei'istics as the 
oleic soap, but to an e\'en greater extent. 'I'rimetliylaniine was con- 
stantly given off in large (luantities, and the onl>' way in which it could 
be handled as a soap was in a mixture of alcohol and wafer in a tightly 
stoppered bottl(\ 

The mixed solubl(> and ins()lul)le fatty acids coml)ined with trimethyl- 
amine showed the same genei'al characteristics as the l)utyric and stearic 
combinations, respectively. 

The instability of the combinations of fatty acid and trimethylamine 
can undoul)tedly be accounlc^d foi' l)y the fact that they are addition 
products in which the tri\alent nitrogen of th(> latter substance cb.anges 
to the pentavalent condition in the presence of an acid. The fact that 
these are weak acids with relatively large molecules is probably also 
significant. It was observed that the hydrochloride was more stable 
than the above salts, and that the sulfate was even more stable than 
the liydrochloride. Tliis instabihty M the fatty-acid combinations and 
tlieii- iT'action in the presence of sodivun chloride may have an imi)or- 
tant bearing on the relation of trimethylamine to fisliiness in butter, 
and may ho of particular significance in explaining why the flavor is 
usually foimd in salted butter. As 3'et, however, the relationsliip is 
not clear. 



The Lecithin Content of Butter 113 

effect of workinci trimethylamine salts of the fatty 
acids into butter 
Tho character of the ii-inu'lliylaminc salts of tli(> fatty acids in pure 
state seemed to justify t!ie following series of experiments, in which these 
salts are incorporated into various typ(^s of ])utier for the purpose of 
determining tiic possibility of their producing the fishy flavor in the 
presence of butterfat. In vi(>w of the d(>sirability of incorporating the 
trimethylamine in logical amounts, tlu> following plan was adoi)t(Hl: 

The largest quantity of lecithin reported in cow's milk by Nei'king 
and Haensel (1908) was used as th(^ rxisis of calculation. These authors 
report 0.1 IG per cent as th(; largest amount found in seventeen samples. 
For the calculations of this experiment, this was assumed to be lecithin 
of the dist(>aryl type, and it was further assuuKHl to be pure lecithin with 
the empirical fornmla assigiunl to the type named. Granting these 
assumptions, this amount would yield on complete decomposition the 
equivak'nt of 8o i):irts pvr million of trimethylamine. This substance 
alone or in acid combination was therefore added to cream, wash water, 
or butt(>r on this basis. It is very evident that because^ of the volatihty 
of some of tlu> materials and because of mechanical loss, none of the 
samples of butter wh(m completed would contain 85 parts per million 
of trimethylamine. The method of arriving at the (juantity to be added 
seemed to afford a uniform basis and to a])pi-oximate in a logical manner 
the amount of this substance^ that might be i)r()duced in butter. When 
the fatty acids wei-e usetl alone; they were added in {]uantiti(>s equivalent 
to the amounts added in the corresponding trimc^thylamine salts. The 
addition of the acids was merely for the purpose of checking against the 
trimethylamine. 

In tabl(>s 3 to 9 inclusive are shown the comments of various judges 
on diffei-ent types of butter containing trimethylamine addc^l as already 
indicated and iMcor])orated by various means. Because of the great 
importance of the personal factor in judging butter, an effort was made 
in all cases to get a nunibca- of men familiar with the various flavor defects 
of the product. In all cas(>s tlie samples were so labeled that the judges 
had no knowledge of th(>ir contents. They were instructed to comment 
on the flavor and to work independently of one another, and it is believed 
that this injunction was can-i(Hl out. In presenting the results in tabular 
form the autho)- has intentionally omitted comments having no direct 
bearing on the fishy flavor. 



114 



George Cornell Supplee 



TABLE 3. Effect on the Flavor of Buttek, of Adding Tkimethylamine and Fatty 
Acids to Raw Sweet Cream with 0.23 per Cent Acid at the Bate of 85 Parts 
per Million of the Former 

{S indicates salted butter) 



Sample 


Material added 


Coinnients by judges 




No. 1 


No. 2 


No. 3 


No. 4 


No. 5 


No. 6 


AS 


Nothing . 








Oily 


Oilv 
Oily 




A 


Nothiiis 




Oily 
I'ishy 






1 AS 


Trinietlivlaiiiinc . . 


Oilv 
I-ishy 




i'ishy 


Fishy 


1 A 


Trimethylamine 

Triincthvlaniinc lactate 


2 AS 












2 A 


Triinetliylatiiiue lactate 

Triinethylamine liutyratc 

Tritncthylaiiiiiie butyrate 

Triiiicth>larninc oleate 


Fisiiy 

!"isliv 
i'ishy 

I'ishy 


( )ily 

l-'ishv 
Oily 


I'i.shy 
iMshy 


I'ishy 






3 AS 






3 A 

4 AS 


I'isliy 
I'ishy 






4 A 










5 AS 


Trinietliylamirie stcarate 

TriincMiylainine stearate 

TriiiiethylainiiK' aiul sohihle fatly 
acids 


i'ishv 
fisiiy 












5 A 












6 AS 




I'ishy 
Oily 

I'isliy 


I'lsliy 




Fishy 


6 A 


Triinctliylaniiiic and solublf fatty 
iicids 


Fish\- 


l<ishy 

Fishy 
Oily 


7 AS 


Triinctliylaniine and insoluble 
fattv acids 


i'"ishy 




Fishy 
Oily 


7 A 


Triin(»thylaniine ami insoluble 
fatt\' acids 




8 AS 


Lactic acid 






Fish>- 




8 A 


Lactic acid 

Butyric acid 

Butyric acid 


FLshy 

I'ishv 
I'ishy 


Oily 








9 AS 










9 A 






Oily 
Fishy 






10 AS 


( )i(Mc acid 


I'isliy 


I'ishy 






10 A 










11 AS 


Solul)ie fattv acids 














11 A 


Soluljle fatty acids 














12 AS 
















12 A 


Insolultlc fattv acids 































The Lecithin Content of Butter 



115 



TABLE 4. Effect on the Flavor of Butter, of Adding Trimethylamine and Fatty 
Acids to Raw Sweet Cream with 0.23 per Cent Acid at the Rate of 85 Parts per 
Million and Then Washing the Butter in Water Containing the Same Concen- 
tration OF the Various Substances 

(S indicates salted butter) 



Sample 


Material added 


Comments by judges 




No. 1 


No. 2 


No. 3 


No. 4 


No. 5 


No 6 


BS 


Nothing 








Oily 


Oily 
Oily 

Oily 

Fishy 

Oily 




B 


Nothing 




Oily 
Fishy 






1 BS 


Trimethylamine 




Fishy 

Fishy 
Fishy 


Fishy 

Fishy 
Fishy 


Fishy 


1 B 


Trimethylamine 




2BS 


Triraethvlamine lactate 




Oily 
Oily 

Oily 


Fishy 


2B 


Trimethylamine lactate 

Trimethylamine butyrate 

Trimethylamine butyrate 

Trimethylamine oleate 


Fishy 

I'ishy 
Fishy 

I'Mshv 
Fishy 

l-'ishy 
Fishy 


3BS 




Fishy 


3B 


Fishy 
Fishy 




Fishy 


4BS 






4B 


Trimethylamine oleate 








Fishy 

Fishy 
Fishy 


5BS 
5B 


Trimothyiamine stearate 

Trimethylamine stearate 

Trimethylamine and soluble fatty 
acids 


Oily 


Fishy 


Fishy 
Fishy 

Fishy 


Oily 


GBS 


Fishy 
Fi.shy 

Fishy 

Fishy 

Fishy 
Oily 


Fishy 




Fishy 
Fishy 

Fishy 


GB 


Trimethylamine and soluble fatty 
acids 


7BS 


Trimethylamine and insoluble 


Oily 


Fishy 


Fishy 
Fishy 




7B 


Trimethylamine and insoluble 
fattv acids 


8BS 


Lactic acid 

Lactic acid 

Butyric acid .... 










8 B 












9 BS 






Oily 
Oily 

I'Mshy 






9 B 






I'ishy 
Oily 


Fishy 






10 BS 


Oleic acid 








10 B 


Oleic acid 








11 BS 














11 B 


Soluble fatty acids 














12 BS 


Insoluble fattj' acids 














12 B 

































116 



George Cornell Supplee 



TABLE 5. Effect ON the Flavor of Butteh, of Adding Trimethylamine and Fatty 
Acids to Raw Sweet Cream with 0.23 per Cent Acid at the ]{ate of 85 Parts per 
Million and Then Working the Substances Directly into the Butter at the 
Same Rate 

(S indicates salted butter) 



Sample 



CS 

c 

1 CS 

1 c 

2CS 
2C 

8CS 
3C 

4CS 
4C 

r)Cs 

5 C 

()CS 

«)C 

7CS 
7C 



8CS 
8C 

«» C^ 
9C 

IOCS 

10 C 

11 CS 

11 c 

12 CS 
12 C 



Material added 



Nothing 
Nothint! 



Triinethylainiiio. 
Trimethvlainiue. 



Triinethylatiiine lactat(^ . . 
Triinethylaniiue lactate . . 

Triinethylainine hufyraie 
Triiiiethvlainitie Inityrate 



Triinethylainine oleate 

Trinu'thylaminc oleate 

Triinethylainine stearate 

Triinethylainine stearate 

Triinethvlamine and sohible fatty 
acids." 

Triinethylainine and soluble fatty 
acids 

Triinethylaniiue and insoluble 
fatty acids 

Triinethylainine and insoluble 
fatt.v acids 



Lactic acid . 
Lactic acid . 



Butyric acid . 
Butvric acid . 



Oleic acid . 
Oleic acid . 



Soluble fatty acids 
Soluble fattv acids 



Lisolnbie fat.ty aeids 
Iusolu!)le fattv ticids 



Comments by judges 



Xo. 1 



I'ishv 



I'ishv 
I'ishy 

Fishv 
Kishv 



Kishv 



l-'ishv 
Fishv 



■'ishy 
■ishv 



Fish 
Oilv 



Fishy 
Fisiiv 



Oilv 
Oily 



No. 2 



Oily 

Fi.shv 
l-'ishy 

I'ishv 
Fishy 

Fishv 
l-'i.shv 



Fishv 



l''ishv 



Xo. 3 



l"i.shv 



Fishv 
iMshy 

I'ishv 

I'ishy 

I'ishy 
I'ishv 



Fishy 
Fishy 

Mshv 



Fisliv 



I'lshy 
Fishv 



Xo. 4 No. 5 



Oily 



l-'ishy 
Fishy 



iMshy 
I'ishv 



Fishv 
Fishy 

Fishy 
Fishy 



Fishy 
F'ishv 



Fishy 

Fishv 
I'^ishv 



Oily 
Oily 

F'ishy 
Fishy 

Fishy 
Fishy 



I'ishv' 



Fishy 

iMshy 
Fishy 

Fishy 
Fishy 



Oily 



No. 



Fishy 
Fishy 

Fishy 
Fishy 

Fishv 
Fishy 



Fishy 
Fishy 



Fishy 
Fishy 



The Lecithin Content of Butter 



117 



TABLE 6. Effect on the Flavor of Butter Made from Pasteurized Ripened 
Cream with 0.32 per Cent Acid, Working Trimethylamine and Fatty Acids 
Directly into the Butter at the Rate of So Parts per Million 

(S indicates salted butter) 



Sample 


Material added 




Comments by judg 


3S 






Xo. 1 


No. 2 


No. 3 


Xo. 4 


Xo. 5 


No. 6 


DS 


Nothing 














D 


Nothing; 














1 DS 


Trimethvlaniine 




Fishy 


Fishy 
Fishy 


I'ishy 
r'ishy 


Fishy 
Fishy 




1 D 


Trimethylamine 






2DS 


Trimethylamine lactate 








2D 


Trimethylamine lactate 














3DS 
3 D 


Trimethylamine butyrate 

Trimethylamine butvrate 


Oily 


I'ishv 
Fishy 




Oily 
Oily 


Fishy 
Fishy 


Fishy 


4DS 


Trimethylamine olcate 






4D 


Trimethvlaniine oleate 














5 DS 


Trimethylamirie stearate 








I'isliv 
Oily 


Fishy 
I'"ishy 




5 D 


Trimethylamine stearate 

Trimethylamine and soluble fatty 
acids . 


Oily 








6 DS 




Fishy 




6D 


Trimethylamine and soluble fatty 
acids 












7 DS 


Trimethylamine and insoluble 




Fishy 










7 D 


Trimethylamine and insoluble 
fatty acids 




l''ish\- 










8 DS 




Oily 










8 D 














9 DS 


Butvric acid 


Fishy 


Fishy 
Fishy 










9 D 


ButjTic acid 










10 DS 


Oleic acid 




Fishy 
Fishy 








10 D 














11 DS 
















11 D 
















12 DS 


Insoluble fatty acids 


Oilv 
Oily 








Oily 
Oily 




12 D 
















1 





118 



George Cornell Supplee 



TABLE 7. Effect on the Flavor of Butter Made from Pasteurized Sweet Cream 
WITH 0.16 per Cent Acid, Working Trimethylamine and Fatty Acids Directly 
INTO THE Butter at the Rate of 85 Parts per Million 

{S indicates salted butter) 



Sample 


Material added 




Comments 


by judges 




No. 1 


No. 2 


No. 3 


No. 4 


No. 5 


No. 6 


ES 


Nothing 














E 


Nothing . 














1 ES 
1 E 


Trimethylamine 

Trimethylamine 

Trimethylamine lactate 

Trimethylamine lactate 


Fishy 
Fishy 

Fishy 


Fishv 
FishV 

I-'ishv 
Fishy 

Fishy 
I'ishy 

I'ishv 
I'ishy 

Fishv 
Oily" 

Fishy 
Fishy 

I'ishy 

Fishy 

I'lshv 
Fishy 


Fishy 


I'^ishy 




Fishy 
I-'ishy 

Fishy 
Fishv 


2ES 
2 E 


Fishy 


I'ishy 




3ES 
3 E 


Trimethylamine butyrato 

Trimethvlamine butyrate 


Fishy 


I''ishy 


Fishy 


Fishy 


Fishy 
Fishy 


4ES 


Trimethylamine oleate 


Fishy 








4 E 


Fishy 

Fishy 
I'ishy 


Fishy 
Fishy 

Fishy 
Fishy 


Fishy 
Fishy 


Fishy 

Fishy 
Fishv 


5ES 


Trimethylamine stearate . . . 




5 E 


Trimethylamine stearate 




6ES 
6E 


Trimethylamine and soluble fatty 
acids 

Trimethylamine and soluble fatty 
acids 


Fishy 


P'ishy 


7ES 


Trimethylamine and insoluble 
fatty acids 

Trimethylamine and insoluble 
f attv acids ... 


Fishy 


l''ishy 
Fishy 




7E 








8 ES 


Lactic acid 










8E 


Lactic acid . . 












9 ES 


Butyric acid 


Fishy 
Fishy 


Fishv 
Fishy 






Fishy 


9E 


Butvric acid 


Fishy 

Fishy 
F^ishy 

Fishy 


Fishy 




10 ES 


Oleic acid 




10 E 


Oleic acid 












11 ES 


Soluble fattv aciils ... .... 












11 E 










Fishy 




12 ES 


Insoluble fatty acids 


Oily 
Oily 








Oily 


12 E 


Insoluble fatty acids . . 



























The Lecithin Content of Butter 



119 



TABLE 8. Effect on the Flavor of Butter Made from Pasteurized Ripened Cream 
WITH 0.38 PER Cent Acid, Working Trimethyl amine and Fatty Acids Directly 
INTO the Butter at the Rate of 40 Parts per Million 

(S indicates salted butter) 



Sample 


Material added 


Comments by judges 




No. 1 


No. 2 


No. 3 


No. 4 


FS 


Nothing 










F 


Nothing 










1 FS 


Trimethvlamine 


Oily 
Oily 

Fishy 


Oily 
Fishy 

Fishy 

Fishy 
Fishy 


Fishv 
Oily' 

Fishy 




1 F 


Triniethvlaruine 




2FS 


Trimethvlamine lactate 




2F 


Trimethjlaminc lactate 




3FS 


Trimethvlamine butjTate 


Fishy 


Fishy 
Fishy 


Fishy 
Fishy 


3F 


Trimethvlamine butjTate 


4FS 


Trimethylamine oloate 




4F 


Trimethylamine oleate 


Oily 








5FS 


Trimethylamine stearate 








5F 


Trimethvlamine stearate 










6FS 


Trimethvlamine and soluble fatty acids .... 






Fishy 




6F 


Trimethylamine and soluble fatty acids 

Trimethylamine and insoluble fatty acids. . . 


Oily 




Fishy 


7FS 








7F 


Trimethylamine and insoluble fattv acids. . . 








Fishy 


8FS 










8F 


Lactic acid 










9FS 


Butyric acid 










9F 


Butyric acid 










10 FS 


Oleic acid 










10 F 


Oleic acid 























120 



George Cornell Supplee 



TABLE 9. Effect on the I'Yavor ok Btttkr NLvue from Pasteurized Ripenkd 
Cream with 0.2S per Cent Acid, of Wohkinc Tkimethylamine and Fatty Acids 
Directly into the Butter at the Rate of 40 Parts per Million 

(S indicates salteil butter) 



Sample 


GS 


G 


IGS 


IG 


2GS 


2G 


3GS 


3G 


4GS 


4G 


5GS 


5G 


6GS 


6G 


7GS 


7G 


8 GS 


8G 


9GS 


9G 


10 GS 


10 G 



Material added 



Notliins- 
^'otllin{^. 



Triniethylaiiiine. 
TrimethyUyuiiie . 



Trimethylaniiiu' lactate , 
Triinethvlaiiiiue lactate . 



Trimetb ylainine butyrate . 
Triinethvlaiiiiue l)utyrate . 



Triiiif'thylainiMe oleate 
Triinethvlamine oleate 



Triiricthylainine stearate , 
Trimethvlaiiiine stearate . 



Triinetliylainino and soluble fatty .acids . 
Trinietiivlainine and soUiljle fattv acids. 



Triinetl!>laniin(! suid insoluble! fatty acids 
Triinetlivlaniine and insoluiile fattv acids 



Lactic acid 
Lactic acid 



Butyric acid , 
Butvric acid . 



Oleic acid 
Oleic acid . 



Comments by judges 



No. 1 



I'ishy 



Fishy 

l''ishy 
I'^ishy 



Oily 



No. 2 



I'ishv 



iMsliy 



llshv 
l-'ishv 



No. 3 



I'^ishy 



Fishy 



Fishy 
Fishy 

Oily 



J'ishy 
Fishy 



No. 4 



Fishy 



Fishy 



All of the samples of l)utter I'opi'osoiitod in. tables 3 to 9 inclusive were 
scored from three to five days aftcn- makiiic;. They were then placed 
in storage for di{^"er(>nt lenjiths of time and rescoi-ed l)y two or more 
judges. The results of tliis examination are shown in table 10. In 
this table are listcMl only those samples showing a fishy flavor by unani- 
mous opinion of the persons judging them. 



The Lecithin Content of Butter 



121 



TABLE 10. Presence of Fishy Flavor after Stor.^ge, in Butter to Which 
Trimethylamine Had Been Added at the Time of Making 



Sample 


Material added 


Age 

(days) 


Comments by judges 


At time of 
making 


After storage 


3 CS 


Trimethylamine butvrate 


40 

267 
20(5 

2m 

243 
243 
213 


Fishy 

Fishv, oily 

Fishy 

Fishy 

Fishy, oily 

Fishy 

Fishy 


Fishy 

Fishy 

Fishy 

Fishy 

Fishy, tallowy 

Fishy 

Fishy 


3 DS 

3 E8 
6ES 
1 FS 


Trimethylamine butyrate 

Trimethylamine butyrate 

Trimethylamine and soluble fattj' acids. 
Trimethylamine 


3 FS 
3 GS 


Trimethylamine butyrate 

Trimethylamine butyrate 









The results obtained from these experiments bring out some very 
interesting faets. ^^'llil(^ there are several conflieting opinions as to 
the presence of the; fishy flavor in any particular sample, it is neverthe- 
less evid(>nt that the greatc^st number of j)ositive comments is found in 
the sami)les containing trinu^thylamine in one form or another. It will 
also be noticed that usually the greatest uniformity of such comments 
is found in the samples containing trimethylamine in unstable form. 
This is particularly true as to the sam])les to which trimethylamine was 
added alone, in combination with butyric acid, oi- in combination with 
the mi.xed soluble fatty acids of butter. These results are in harmony 
with the volatility, the taste, and \hv odor of tlie compounds in pure 
state. The lack, in a few instances, of a majority opinion with regard 
to the samples containing trimethylamine oieate, trimethylamine stearate, 
and trimethjdamine with tlu> mixed insoluble acids, might b(^ explained 
on the basis that, since these substances were so extremely unstable, the 
ti'imethjdamine had nearly all volatilized before the time of scoring. 
Th(^ gi'(>ater number of positive connnents from the salted- butter is also 
worthy of note, and, from what is gi>nerally known regarding the occur- 
rence of the fishy flavor in .•^uch butter, it might tend to strengthen the 
trimethylamine theory (jf this flavor. Another feature found in this 
series of experiments is that a greater number of fishy-flavored samples 
were found where the acidity of the cr(\-un was the lowest. This condi- 
tion is in harmony with the chemistry involved, for the reason that butter 
made from low-acid crcnun contains less lactic acid when fresh than is 



122 George Cornell Supplee 

found in butter made from lii<>;li-a('id cream. It is thei'(>fore conceivable 
that trimethylamine given off by the unstalile compounds which were 
added could not be taken up by the excess lactic acid in the butter to 
form the more stable trimethylamin(> lactate. The findinfj; of numerous 
fishy-flavored sami)les where trimethylamine lactate had been added 
to low-acid butter mij>;ht be explained on the basis that certain conditions 
were present, possibly particular enzymes, which were capabk^ of bringing; 
about the moi-e ra])id dissociation of the trimethylamine lactate, and 
that due to the absence of sufficient free lactic acid to hold tiie trimethyl- 
amine it passed into the free state. This explanation is bornc^ out by the 
fact that in the buttei' from high-acid cream there werc> a small(>r number 
of sampk'S showing fishiness where the kictat(' was added. It might be 
stated further that one would naturally expect to find a gicatei- variety 
of enzymes capable of bringing about the above-described (k'composition 
in unripened than in i-ipened ci'eam. 

The evi(k'nc(» obtained from the various sam])les of buttei' to whicii 
trimethylamine butyrate was nddcd, indicates a striking relationship 
between this substance and the fish\' flavor. This seems to be true 
reganUess of the type of butter, and to a ceilain extent regai'dless of 
the presence of sodium chloride. The (>xtremely \olatile natui'e of this 
substance and its ciiai'actei'istic odor in pui'e statt^ easily account for 
the results obtained. The fact tliat Ihi'.i'e were more positive conunents 
on the butters cojitaining l)utyric acid alone than there were on butters 
containing the other acids alone, indicates tiiat this substance may be 
a contributing factoi- in the development of tiie fishy flavor under natural 
conditions. This feature indicates also that tiie fishy flavoi' may be dnv 
to a definite balance between a decomposition yielding trimethylamine 
and one yielding butyric acid, with the conse(iuent foi'mation of trinu^thyl- 
amine butyrate. This associative action would be entirely ])()ssible in 
storage butter, judging from what is known icgarding these f(>rmentations. 

In the foregoing discussion \\\c writer has called attention to certain 
theoretical possibiliti(>s which miglit correlate the findings with what 
is generally known regarding the various types of normal and of fishy- 
flavored ])u1ter. It may be said, however, that the evidence! points 
strongl}' toward trimethylamine as a conti'ibuting factor in fishy-flavored 
butter. The amount of this sul)stanc(^ responsible foi' the flavor described 
as fishy in these ex[)eriments is very small. It would be a hazardous 



The Lecithix Content of Butter 123 

guess to assign a definite quantity, other than to say that in all cases 
there was less than 85 parts per milhon. 

QUANTITATI\T] ESTIMATION OF TRIMETHYLAMINE IN FISHY BUTTER 

The results obtained by working trimethylaniine into normal butter 
warranted an attempt to isolate this substance from samples of fishy- 
flavored butter found on the market. One of the first difficulties met 
with in this conn(H'tion was the lack of a method which would accurately 
measure the small amounts of trimethylaniine that would be found. 
The method that was finally woiked out consisted of a combination 
and modification of the methods of P'olin and Macallum (1912) for 
ammonia and of Budai [Bauerl (1913) for trimethylamine. The adap- 
tation of these methods for this pu!i)ose was as follows: 

The material in which trimethylamine and ammonia were to be deter- 
mined was concentrated to a volume^ not exceeding 15 cubic centimeters. 
This material was then i)laced in the pi'oper tube of the Folin apparatus, 
10 grams of anhydrous potassium carbonate was added, and the mixture 
was covered with a thin layer of kerosene to prevent foaming. This 
mixture was aspirated for fiv(^ hours. The ammonia and trimethylamine 
set free by the potassium caibonate was collected in N/IO hydrochloric 
acid. The excess acid was titrated with exactly N/100 alkali, methyl 
red being used as the indicator. The results of this titration gave the 
total amount of the mixture of ammonia and trimethylamine. To this 
titrated mixture 10 cubic centimeters of a neutral 40-per-cent formalde- 
hyde solution was added. The annnonium chloride present reacted with 
the formaldehyde to foi-m h3'di-ochloric acid and hexa-methylcne-tetramine 
according to the equation 

6 HCOH and 4 NH.CL = (CH2)r,N,, 4 HCL, and 6 H.O. 
The hexa-methylenc-tetramine being neutral, the hydrochloric acid 
liberated from the ammonium chloride was titrated and the ammonia 
was calculated from this titration. Since the trimethylamine hydro- 
chloride present was not affect(>d by the formaldehyde, the trimethyl- 
amine was calculated by differences. Since this is essentially a micro 
method, the technique involved is of the utmost importance. In all 
cases the volume of the solution to l)e titrated was kept as nearly con- 
stant as possible, and the same amount of indicator was used for each 
titration. A check on the standard acid and alkaU was made with each 



124 



George Cornell v^upplee 



determination. ;uk1 llie end-points of all neutralization processes were 
compared colorinietrically with the standard neutral color. In table il 
are shown the i-esuUs ()l)tained by this meth(id from mixtures of known 
amounts of trimelliylamine liydrochloride and annnonium chloride: 

TABLE 11. Ekficiency of the Modified Micro Method for Estimating 
Thimethylamine and Ammonia 



Samplo 


Ai'tual ;uu()unt of 

(CH:ji:iX and XHs as 

liydrocliloride.s 

([uiliip:r;uiis' 


Amount recovered 
(milligrams) 


Percentage 




()(> 

()(j 
2 30 

1 84 
92 
92 
40 
40 
270 
27C) 
27t> 


XH, 


(CIIsl.X 


XHa 


(CH3)3X 


NH3 


1 


(U) 

(•)(■) 
2 30 

1 81 
92 
92 
4C) 
40 
27!') 
27() 
270 

27(i 

1 81 
92 
40 
(il! 
092 


01 

51 
2 25 

1 79 
885 
911 
0.442 
0.47(> 
295 
295 
295 


() 03, 

03 
2 278 

1 820 
035 
935 
47() 
47() 
.323 
297 
297 

289 

1 820 
918 
1.59 
0.040 
105 


99 70 

97 75 

97.83 

97 28 

90.19 

99 35 

90 09 

103 48 

100.88 

100.88 

100.88 


99.55 


2 


99. 55 


3 


99.04 


4 


98 91 




101.03 





101.03 


7 


103 48 


8 


103 48 


9 


117 03 


10 

11 


107.01 
107.01 


12 

13 


104 71 
98 91 


14 

15 




99 78 
99.78 


10 

17 





100.31 
114.13 



The results shown in talkie II having justified the reliability of the 
micro method (oi- nieasuriiiL;; sin;dl amounts of trimethylamine, a number 
of fishy-flavoird samples of butter were subjcH'ted to analysis. The 
})Utter was thoroly washed in a scpai'atory funnel five times with equal 
volumes of water aciditicMl witli hydi-ociiloric acid at the rate of 25 cubic 
centinK>t(M-s of noi-mal acid to the htcv. The wash water was then evapo- 
rated to a small vohime as (juickly as possible, and the trimethylaminc 
and ammonia were determined as outlined al)Ove. 

In table 12 are shown th(^ trimethylaminc and annnonia results obtained 
from fishy butter appearing in comnKM'ce and procured from widely 
different locahties. The annnonia results are shown as a matter of 



The Lecithin Content of Butter 



125 



interest but they probably have no direct bearino; on this particular 
problem. Since the expcM'iuients with artificially produced fishy flavor 
seertied to indicate the importance of acidity, the acid values of the samples 
are also included. The acidity is expressed as cubic centimeters of N/10 
sodium hydroxide used to neutralize 20 grams of butter in boiling alcohol. 
Trimethylamine and anniionia are expn^ssed in parts per million. 

TABl.l'' 12. 'rpaMi.TTTTi. \MTX!o AND Ammon'i.\ Content .^nt) .'\cid Content of 
' -Samples of Fishy-flavored Butter 



Sample 


TriinethylaniinR 
(parts per million) 


Ammonia 
(parts per million) 


Acid 
value 


1 


30 1 
35 4 

2S S 

27 3 

14 

None 

None 

20 

No analysis 

No analysis 

No analysis 


11.2 

14.4 

15 2 

26.1 

18.3 

119 

20 () 

55.0 

No analysis 

No analysis 

No analysis 


3.8 


2 


5.7 


3. . 


5 5 


4 

5 

6 . 


5 4 
6.8 
3 5 


7 


2 7 


8 


3.8 


9 


3.8 


10 

11 


3 
11 



The data submit li'd iii table 12 arc of considerable interest in view 
of the history of some of the saini)les. On arriving al the laboratory, 
all of the samiiles, with the cx('e])ti()n of samples o and 11, were scoi'cd 
as fishy by several judges. These two exceptions wvw samples of butter 
sent from a distance and were i)r(>sumably scored as fishy when shipp(>d 
but could not be so judged when received. It will be noted that in both 
cases there was a higher acid value than in any of th(> other samples, and 
also that th(> ti'imethylaniin(> c()nt(>nt of sample 5 is low. With these 
exc(^|)tions the acid value appears to 1)(> relatively constant, as does the 
trimethylamine content with the excei^tion of samples () and 7, in which no 
trimethylamine whatever was found. Tlu^ majority of these results would 
seem to point to a definite trinuMhylamiiie-acid relationship, as referred 
to elsewhen^ in this pai)er. It is to hr regr(>tted that in three instances 
the sami)l(^ of butter submitted was too small to warrant analysis. The 
available^ data, however, point to trimethylamine as one of the causal 
agents in fisjiy-flavored butter. 



126 



George Cornell Supplee 



DEVELOPMENT OF FLSIIY FLA\"OR IN EXPERIMENTAL BUTTERS 

In oixlcr that the (l(n'('loi:)ni(Mit of th(^ fishy flavor might bo more care- 
fully s1u(li(Ml. throe s(>rios of oxporiuiontal l)uttors wore made with the 
olijoct of determining th(> influence of pasteurization, of acidity developed 
during lipening of the cream, of adding lactic acid to the cream, of inocu- 
lating butter with lactic-acid bacteria, and of salt. The procedure fol- 
IowchI in each of tlu^se series consistiMl of making nine different types of 
butter, salted and unsaltcd, fi-om tlu> same oi-iginal lot of cr(\'un. The 
different series were made at intervals of from three to six weeks. The 
description of (\icli of tlu^ diCfercMit types of butter in each of the series, 
and the designation of the sam|)les, are shown in table i;>: 

TABLI'i I'A. 1)ks<uii>ti()N of Tvi'ks ov Bittku IMadk to Sti uy thk Dkvelopme.nt 

(iK Fishy Fi.a\ou 









Xame o 


' sample 






Treatment, of cream 
or butter 
















Salted 






Pnsalted 






A series 


B series 


C series 

('SP>S 

CSITS 


A s(^ries 


B series 


C scries 


l!a\v sweet cream 

I'asteurized sweet cream . 


ASiJS 
ASILS 


BSI!S 
BSHS 


ASR 

ASH 


BSR 

BSII 


CSR 
CSII 


tlaw cream ripened with 
starter 

rasteurized cream rijiened 
with starter . . . 


A tins 

APRS 
AiiP.XS 


BRIiS 
BPPS 
iUMJXS 


(•i!HS 
Cl'iJS 

c'tnsxs 


AlMl 
ABH 

A[u:x 


bi;r 

BPR 
BURN 


CRR 
CPR 


Raw cream ripened natu- 
rallv 


CRP.N 


Raw sweet cream with Bnc- 














icrium lac'is (icidi worked 
int.o l)utter 


ASRBS 


BSRBS 


CSI{P,S 


ASI!B 


iiSHB 


CSRB 


Pasteurized creatn witli 




Unci lit mil liirlis acnii 
worked into butter 


ASIIBS 


BSIIBS 


rsiiBs 


ASHB 


I'.SIIB 


CS 11 B 


l?aw sweet cream acidifii'i! 
with lactic acid 


ASK'LS 


BSRLS 


CSRLS 


AS1!B 


]',SHL 


CSP.L 


Pasteurized cream aciditievi 
with lactic acid 


ASIILS 


i5sm,s 


CSifLS 


AS;iL 


liSlIL 


CSIIL 



The samples indicated in tabk^ 13 were placed in storage at a tempera- 
tm'C of 0° F. or lower, and were scored by tliree or foiu' judgc^s at various 
intervals. The I'csults of these scorings are gi\"eti ifi table 14. Non- 
characteristic flavors are purposely omitted from tiiis table. 



The Lecithin Context of Butter 



127 



TABLE 14. Comments of Judges on the Different Types of Experimental 
Butters after Various Lengths of Time in Storage at 0° F. 



Sample 



ASR . . . 
ASRS . . 
ASRS . . 
ASRS . . 

BSR 

BSRS . . 
BSRS . . 
CSF>S.. 
CSRS . . 

BSHS.. 
CSH . . . 

csirs.. 

ARR... 
ARRS.. 
ARRS.. 
ARRS.. 
ARRS.. 
BlUiS.. 
CIUIS.. 
CRRS.. 

BPR... 
BPR. .. 
API^S.. 
APRS.. 
BPP.S. . 
BPHS.. 
BPRS. . 
CPRS.. 

ARRNS 
BRRN . 
BRRXS 
BRRXS 
BRRNS 
CRRXS 

ASRB.. 
ASRBS. 
ASRBS. 
BSim.. 
CSliBS. 
CSHBS. 
CSRBS. 



Acid 

in 

cream 

(per cent) 



27 
0.27 
0.27 
27 
IS 
IS 
O.IS 
0.1(52 
0.102 

0.10 

0.111 

0.111 

O.C.S 
0.()S 
0.()S 
O.OS 

(IS 
o.r)()7 
o.r)(i7 
o.r)07 

:)2 

.J2 
O.C)() 
0.()() 
52 

o.r)2 
o.r)2 

0.5(12 

0.()75 
57() 
0.57() 
57!') 
57(1 
O.dlK) 

27 
27 
27 
IS 
1(12 
0.1(12 
0.102 



Days 

in 

storage 



45 
45 

180 

285 
45 
45 

285 
45 

285 

l,*) 

i:!() 

45 

45 

45 

90 

130 

285 

45 

(10 

285 

45 
l.'iO 
130 
285 

■15 
130 
285 

<)0 

130 
45 
45 

00 

285 
00 

45 
45 

130 
45 
45 
90 

285 



No. 1 



Fishy 
I'ishy 
Fishv 



Oily 
Fishv 



Metallic 
^ietaliic 



MetaWic 
MetaUic 

Metallic' 
:\letallic 



lishy 
^ietallic' 

Metaliic 
Oilv 



Oily, fishy 
Fishv 
Fishv 
Fishy 

Fishy 
Fishv 
Fishy 

Oilv, fishy 



Comments by judges 



No. 



Fishy 



Fishv 



Oilv 



Metallic 

Aletaliic 

( )ily 

Metallic 

Meoallic 

Metallic 

Oilv 



Fishy 

MetalUc 

Metallic 



Oilv 



I-4shv 
I'i.shv 



Fishy 
Fishy 
Oily * 



No. 3 



I'ishv 

Oily 

Oily 



14shy 
Metallic 



Metallic 
Oilv, fishv 
Mx'tallic 
Fishy 

^ietaliic 
MetalHc 

Oily 

M(!tallic 



Oilv 



Fishv 
Oily 



Oily, fishy 
I'4shv 
Oily,' fishy 



Oilv 



Oily 
Metallic 



No. 4 



Fishy 
Fishv 



Fishy 



Fishv 



Oily 



128 



George Cornell Supplee 

TABLE 14 icondudi-d) 



Sample 


Acid 

in 

creiini 

(per cent 1 


Days 

in 

storage 


Comments by judges 


No. 1 


No. 2 


No. 3 


No. 4 


ASHBS 


189 
0.1 S9 
141 
141 

(i^S 

(i:?s 

(l.'iS 

(•>;^s 

(Km 
(Km 
()()().■) 
472 
472 

(;'.)() 

(i'.K) 
-^\)\ 
' ;■■;•. 11 
-)04 
n{)[ 
501 


130 

28.-) 

«)() 

4.-) 
00 
i:50 

2s:) 

4.-, 
00 

2sr, 

285 

130 
285 
15 
90 
45 
45 
285 


Fisliy 

M eta' lie' 
Metallic 

I'ishy 


Oily 






ASHBS 


Fishy 
Metallic 


Metallic 


CSHBS 


Metallic 




CSHBS 




ASRLS 








ASRLS 




Fishy 
Fishy 
Fisliy 




ASRLS 




I'^ishv 

I'^ishv 

I'ishv 

Fishv 

Fishv 

Metallic 

Metallic 

Metallic 
I-ishv 
Oilv 
I'isliv 




ASRLS 

BSRLS 


iMshy 


r'ishy 


B.SRLS 








BSRLS . 




Fishy 


Metallic 


CSRLS 






CSRLS 




Fishy 

Metallic 
Fishy 


Oily 


ASIILS 






ASHLR 






BSIILS 


I'ishy 




BSHLS 






CSHL 


I'ishy 
Fishy 






CSIILS 








CSIILS 




I'-isliv 


Oily 













In ('onsidcrliii;- the r('.'^ul!.-< from the (liffcnMit tyix's of c>x])('riin('ntal 
butler, it is evident that there is consideral)!'' diversity of opinion among 
the judges as to the ])resenee or i\\c absence of th(^ fisliy flavor in certain 
sanij)les. It is also (>vident I hat there is some i-elationsliip between the 
metallic, oily, and fishy flavoi-s, jjarticnlarly wIkmi these flavors are not 
sufficiently pronounced to l")e distinctive as was the case in those samples. 
This would secnn to indicate^ that there are jMOssibly certain fundamental 
conditions which are common to lh(> development of (^ich of these flavors. 

Even tho there is ditf(>rence of opinion as to the presence of the char- 
acteristic flavors, certain conclusions may be diawn from these experi- 
ments. Probably one of the most significant is the presence of the fishy 
or the metallic flavor in thc^ salted iMitters. Of a total of 105 character- 
istic comments, 93 are found in the samples containing salt. Another 
conclusion which may be drawn from tlie relative agreeuKnit of the 
judges, is that tlie fishy fiavor apix'ars oftener in the butter made from 



The Lectthix Content of Butter 129 

hisli-acid croam tluui in thai from low -acid cream, there being little 
difT(M-ence whether the acid was developed l)v the use of starter, by ripen- 
in<i- naturally, oi- by the addition of lactic acid to raw sweet cream. 

These findings, compared with the results ol)tained from pasteurized 
cream either churned sweet, ripened with start(M', or acidified with lactic 
acid — all of which showed f(nver fishy samples llian did raw cream — cleai'ly 
indicate that the fiuuiamental cause of this buttiM- defe(;t is primarily 
biological, not bi-ought about by a spontaneous ch(>mical change in which 
su('h agencies do not play a i)ai-t. While it is evident that acid plays 
an important role in the developnuMit of the fishy flavor, it is equally 
clear that there are ()th(>r important contributing factors. Just what 
these factoi'S are, is unknown. Tlu^ variable results obtained from the 
same tyjx> of I)utter in the different s(M-ies would indicate* that the original 
cream or milk possessenl the unknown factors which in the presence of 
lactic acid deteiinincMl the dev(>lopment of the flavor. Fi'om the fact 
that pasteurization tends to re(luce the occurn^ice of tiie fishy flavor, it 
is quite probal)le that these; agencies arc bacterial enzym(>s which are 
only partially inactivated by heat ; oi- it may even be possible that certain 
microorganisms wliich nic incoi-poi'ated in the butt(M' fi'om the cream, 
either in a living or in a dead condition, could on autolysis liberate the 
enzymes capal)le of supi)!ying the detcM'inining factor. It may also be 
added that pasteurization may kill certa.in (Mizymes and not others, the 
particular ones that are inifiortant l)eing among those killed. 

These contentions are fui'thei' supported by the fact that in the butters 
made from raw sweet cream tluM-e is a sugge^stion of fishiness after the 
first storage period which is not found after the longer periods, the dis- 
appearance or lack of furthei- dc\'(dopmeiit of the flavor l)eing due to the 
absence of tlu* pr()[)(M- acid condition. It is clear that large numbers of 
Bdcteriuni lacfis ac/'fJi added dii'CH'tly to l)Utter without their usual accom- 
panying by-products are not the cause of any characteristic change in 
flavor. 

VAUIATIOX IX ACID VALUE OF EXI'ERIMEXTAL BUTTERS 

The importanc(> of acidity in the manifestation of the fishy flavor by 
trimethylamine, and the i-idativ(dy constant acid value of the miscel- 
laneous samples of fishy buttei- found on the market, emphasized the 
importance of studying this factor in the experimental butters described 



130 



George Cornell Supplee 



above. The variation in acid value of the different types of butter in 
each of the three series is shown in tables 15, IG, and 17. Results are 
expressed as cubic centini(4ers of N 10 alkali necessary to neutralize 
20 grams of l)utler in boilin"; neutral alcohol. 



TABLE 15. Variation in Acid Value of Experimental Butters of A Series 
AFTER Various Storage Periods 



Sample 


Acid value after various storage 


periods 




4.3 days 


86 days 


128 days 


310 days 


ASR 


8.8 
7.4 

5.8 

5.7 

8 2 

8.7 

7.7 
8.1 

8.9 

8,8 

9 7 
7.4 

5 7 

5,8 

8 9 
8 9 

7 
7 4 


10 5 

8 

8.6 
5 9 

8.5 
8 4 

7.9 

7.S 

8 () 
8 4 

10 4 

7 , 7 

8 6 

5 9 

8,8 
8.9 

7 1 

6 5 


10 8 

8,2 

10 

5,7 

9 ;^, 

8,6 

8.2 

8 

9 2 

8.7 

11.2 

7,8 

9.5 

5.8 

8.8 
9.0 

7.2 
7.1 


13 


ASKS 


10 


ASH 


12 1 


ASUS . 


7 2 


ARl! . . 


10 3 


AKRS 


10 3 


APR 


9 8 


APRS 


9 6 


ARRX 


10 5 


ARRNS 


10 5 


ASRB 


15 


ASRBS 


9.8 


ASHB 

ASHBS 


14 3 

9 7 


ASRL 


10.3 


ASRLS 


10 


ASHL 


8.0 


ASHLS 


8.5 



The (lata presented in tabl(>s 15, IG, and 17 show many interesting 
featuivs, some of whicli are wortiiy of discussion in connection with this 
pi'cbl(>m. It may be stated in the Ix'oinning that the variations in acid 
value of the different types of butter i)oint to biological agencies as the 
cause of those variations. The lower acid value obtained in nearly all 
instances from salted l)utter indicates a pi'cscrNative action by the salt, 
a function wliich is well known. The greatest increase in acidity is 



The Lecithin Content of Butter 



131 



shown in the butter made from raw sweet cream. It is interesting to 
note that very few of the samples were scored as fishy. When such a 
condition was suggested, it is to be noted that it occurred after the first 
storage period, when the acid vahie was lowest. 

TABLE IG. Variation in Acid Value of Experimental Butters of B Series 
AFTER Various Storage Periods 



Sample 


Acid value after v-arious stora.!?e 


periods 




20 days 


88 days 


126 days 


286 days 


BSR 

BSRS 


13 9 

9.7 

7 3 
G 3 

10 1 
10 1 

10 1 

9.8 

11.3 

11 2 

13 6 
10. G 

8 1 

G.8 

11.5 
11 5 

9 4 
9.5 


14. G 
9 5 

9 
G 5 

10 2 
10.1 

10 1 

10 1 

11 7 
11.7 

13 5 

10 8 

8 4 
G.G 

11 5 
11.1 

9.4 
9.G 


15 2 
10 

10 6 

G.S 

10 8 
10 4 

10 
9 G 

12 G 

11 8 

14 3 

10 8 

9 

6.8 

11.6 
11.6 

9.5 
9 6 


17.5 
10 6 


BSH 


13 4 


BSHS 


7 6 


BRR 

ERRS 


11.9 
11 3 


BPR 


11 2 


BPRS 


11 


BRRN 

BRRNB 


14 3 
13 8 


BSRB 


15 4 


BSRBS 


12 


BSHB 


9 2 


BSHBS 


8 


BSRL 


12 9 


BSRLS 


12 6 


BSHL 


11 


BSHLS 


10 1 







With reference to the samples from pasteurized cream as compared 
with those fiom raw sweet cream, it will l)c^ noticed that pasteurization 
has tended to cause a lowering of the acid vahu^ l)ut has not entirely 
prevented its gradual increase. This would be in accord with possi- 
bilities already stated regarding bacltMial etizynies. The data show also 
a retarding action exerted by the acid originally in the cream. This is 
evident in the butter made from both raw and pasteurized cream ripened 



132 



(^tEokgk Cornell Supplee 



with startcM', from i'jiw ci-cain ri})('iu'(l iialnriilly, and from both raw and 
pastciii'izcd croam (o which lactic acid has hc(ai added. In coxaparing 
the I'csuhs from these samples it v.iil be obsei'ved thai cream ripened 
naturally shows the greatt^si increase in acid value, raw (a'eam ripened 

TABLE 17. N'akiation iv Acu) Value of E.kperimental Hutteus of T Series 
AFTER \'ar!')i;.s St >'i\';-j Peuiods 



Siirnpk! 



CSR . . . 
CSRH . . 

CSH . . . 
CSHS.. 

CRR. . . 
CRRS. . 

CPR .. 
CPRS. . 

CRRN . 
CRRNS 

CSRB . 
CHRB8. 

C8HB . 
CSHBS. 

CSP.L. 
CSRLS. 

CSHL . 
CSIILS. 



Acid viilue after A'ariou.s storage periods 



G Ja\"s 



4.4 
4.4 

7.() 

7.5 

G.t) 
6.5 

S.5 
S.2 

7.S 
6.1) 

4,4 
4.4 

s:.i 

S.4 

5.8 



4S dav.- 



00 days 



10.4 

7.9 


11.0 

8.0 


7.G 
4.7 


11 
4.4 


S . 2 


10 4 

8.4 


7.5 
7.0 


8.9 
7.0 


9.5 
9.0 


10.0 
9.1 


10.2 

7.4 


11.8 

7.9 


0.5 
4.4 


7.7 
4.4 


8.1 
8.5 


8.0 
8.4 


GO 

5 . 7 


5.9 
5.G 



272 davs 



13 


5 


9 


7 


12 





5 


2 


12 


8 


10 


10 


8 


2 


12 


9 


11 


3 


13 


5 


9 



7 1 



9.8 
9 5 

G.7 
6.3 



with starter a little less in»Te;ise, pasteurized cream ripened with starter 
a still less increase, an.d cream to which lactic acid has been added 
the least incr(>ase of any in the grouj). The low acid \'alue caused by 
the addition of lactic acid mia;hlt i)Ossib]y be ex])lained in one of two ways: 
either the addition of the acid in ])ure form has tended to inactivate the 
enz^^mes, oi- the lactic aciTl rc^aintn! in the butter lias been changed to 
butyric acid during stoi-age — which is entirely possibl(> by enzymatic 



The IjEcithin Content of Bittter 133 

action. If such a chan^c^ as tli(> latter did take jilaee, a lowonn"- of the 
acid valiu^ would l)o manif(>sted IxH-ausc of the formation of a weaker 
acid which pro))al)ly has resulted from the splittino; and condensation 
of two parts of the stronger' lactic acid. The possibilit}'- of butyric acid 
being formed in this way might })e supportcnl l)y the fact that the other 
samples from high-acid cream showc^l a lower acid value than those 
from sweet cream but had a high(>r ^'alue than those to which pure lactic 
acid was added. Even tho this change^ did take place, however, it is 
improbable that it coukl (nitin^y accomit foi- the low value indicated. 
It is more probable that the lactic acid acts as an inhibiting agent. A 
study of the tabl(\s will show that the incrc^ase in acid value of the sour- 
cream butters seems to be gi-eater when the amount of i)ure lactic acid is 
lowest in the cream. In using the phi-ase " ])ure lactic acid," rc^fei-ence 
is made to tliat which was added and also to that developed by bacteria, 
it being logical to assunK> that tli(> gr(>atest amount so devc^loped is found 
in pasteurized cr(\'un ripcnied with starter and the least amount in the 
raw cream rii)ened naturally. The condition mentioned above also sup- 
ports the theoiT of devitalized enzymi^s, alt ho it is moi-e difhcult to explain 
why approximately the same degree of connnercial lactic acid has a more 
marked effect than the acid produciMJ by bacteria. It would seem that 
the structure of the particular lactic acids involved produced different 
results in this r(>sp(>ct, or that the other acids ])roduccHl by the bacteria 
are less.inhil)itive than the lactic. 

Regardless of what the (explanation for the vai'iations may be, the data 
seem to indicate that there may be a r(>Iationship bc^twecMi the acid value 
and the fishy flavor, not so nuicli by a constant condition as by a pi'oper 
balanc(e betwcHMi the J)rogr(>ssi^■e develo])nient of the acid value and some 
other contributory caus(>. The most favorable condition would seem to 
be a very gradual increase' in acid value, and one that would be in prop(>r 
harmou}^ and relationship to some other important and transient factor. 
If these views can to any degi'ee .s(»rv(> as a basis of (explanation, it is 
comparatively easy to see how an iiuproper balance of any one of the 
conditions would determin(> the pr(esence oi- the absence of the fishy rlavor. 
It might also be c()nc(Mval)k> that the int(Misily of th(> ti'ue flavor would 
be in invers(e proportion to the degre(e in which thes(> factors wei'e out 
of ('(luilibrium. Such a coiic(e):)tion could explain the occurrence and the 
tlisai)i)earanc(^ of the flavor in the same sampk; of butter at different 



134 



George Cornell Sitpplee 



times, why tlie fishy, the inclnlhc, and th(> oily flavors schmii to bo closely 
related, and possibly \vh\' triniethylamine can Ix^ detected in some fishy 
butters and not in others. 



trlmethylamixe and a.mmoxlv coxtext of experimental butters 
The micro method already described was used to ascertain the ti'i- 
methylamine and annnonia content in the expia'imental butters after 
difl'erent lenjitlis of \i\uv in stoia<ie. The results of these detcM'minations 
are shown in tables IS, l<), and 20. Amounts of the substances are 
expi-essed in parts jxm- million. 

TABLE 18. TiuMKTiiYi,.\MiNK AND .\mmo.\i.v Contknt of Expeki mkvfal Biitteus in a 
Skiuks after \'arious Stou.vck I'kriod.s 



Sample 


After 1 


2S ,!;iy.s 


After 347 days 




(cn,\,N 


XH, 


(C'H.lsX 


NH,, 


ASH 


X\)ne 
Xoiu^ 
Xone 
X'one 
Xone 
3 1 
Xone 
Xoiu> 
X'one 
13 .") 
Xone 
Xone 
Xone 
Xone 
Xone 
X'one 
Xone 


3() 
17 2 

2:; ;', 

2'.; 2 
Id S 
31 
20 7 
2.') :; 
22 4 
IS ' 

22 1 

23 1 

17 ;•. 

14 2 

in :; 

1(1.3 


Xone 

Xone 

Xone 

Xone 

X(>u(! 

0.4 

Noni^ 

X()n(^ 

X( )ne 

Xone 

Xone 

Xone 

11 S 

1.") 2 

(; 

X'one 
Xone 


17 6 


ASI{S 


8 4 


.\SI1. 


10 6 


ASUS 


G 8 


\UU 


30 S 


\iM!S 


18 2 


Al'l! 


24 4 


\P1IS 


24 


arkn 


25 


AUUXS 


6 8 


asi;h 


24 4 


ASIiBS 


12 8 


ASIIB . 


10 8 


ASRL 


9.4 


ASIILS 


10.7 


ASIIL 

\SHLS 


12 2 

8 8 







The I'csults shown in tables IS, 1<1, and 20, alt ho ei'ratic, are of impor- 
tance as indicatinii' the wiriations in decomposition in the same and in 
diffcM'ent lots of cre.am, and fui'ther emphasiz(> ili(> complexity of a problem 
of this nature. The snnie o;en(a-al iv^sults with respect to enzymatic 
;icti\ity in sailed and in unsalted l)Utter from law, pastem'ized, and 
iip(>ned ci'cam ai'c found here as were found in coimection with the acid 
values of the same butters. Alt ho th(> triniethylamine results arc some- 
what discordant, a tendency is shown for the presence of this substance 



The Lecithin Content of Butter 



135 



to harmonize with tli(> samplps scored as fishy, metalKc, or oily. Of 21 
samples in which trimethylamine was found, 15 were assigned one of 
the characteristic flavors by one or more of the judges at some time during 
the storage period. In 5 of the; remaining instances, it is to be noted that, 
while trimethylamine was found in the same type of butter of the same 
series, its presence did not harmonize with the characteristic flavor in 
the salted or the vmsalled sampl(\ On the other hand, there were 10 
samples of different types of l)u(ter whicli wer(> indicated as having a 
characteristic flavor by one or more of the judges at some time during 
the storage period, in which trimethylamine could not be detected. 



TABLE 19. Tkimethylamink and Ammonia Content of Experimental Butters in B 
Series after 323 Days in Storage 



Sample 


(CIl3)3N 


NH3 


BSR 




None 
None 


35 2 


BSRS . 


IS 


BSH 




BSHS 


53 
None 

8.2 
10 G 

9.4 

4.7 
None 
35.4 
None 

5.9 
None 
None 
None 
None 
None 


10 2 


BIU? 


32 8 


BI^RS 


21.0 


BPR 


23.8 


BPllS 


25 


BRRN . . 


27 


BRRNS 


32 2 


BSRB 


29 4 


BSRBS... 


20 


BSHB 


23.0 


BSHBS 


10.4 


BSRL 


23.0 


BSRT.S 


17.0 


BSHL. . 


7.4 


BSHLS 


11.4 







While these results aiv not absoluti^ly coiiclusi\-(\ tii(M'e is ncn^ertheless 
an indicat'ion that trimethylamine may b(> one of the contributing factors 
in the develoj^ment of tlic tru(^ fisiu' flavor. It has been shown that 
this substance is capable of [)roducing a flavor described by butter judges 
as fishy, this l)eing i)articulai'ly true in I he pres(>n('e of butyric acid. 
Furthermore, it has been shown that trim(>thyL-unine may be present in 
tishy-flavored butt(M-. Therefoi'(> it would not ai)pear to be beyond the 



136 



George Cornell Supplee 



realm of possibility that the results shown in this paper point to a definite 
trimethylaniine and acid relationship as l)einji; the cause of that flavor 
in butter which resembles the fla^-or of herrinj;' or mackerel brine, and 
that non-typical flavors n^stunbling other fish pi-oducts, or the metallic 

TABLE 20. TniMKTIlYLAMINK AND AmMONMA CoNTKNT of Kxi'KKIMENTAL BuTTERS IN C 

Skuiks afteu Variocs Stokaoe Periods 



Sample 


After 00 (!a>-s 


After 310 days 




fCHalaX 


XII, 


(CH3)3N 


NH3 


CS1{ 


Xonc 
Xoiu' 
Xonc 
Xonc 
Xonc 
Xonc 
Xoiu^ 
X^oiic 
Xoiu^ 

XolU! 

Xonc 
Xonc 
Xonc 
Xoiu- 
Xonc 
Xon<! 

XolK! 

X'onc 


;;o 2 

20 

:i2 1 

10 7 

;5() :; 
:il 2 
31 7 
•X\2 
;57 
27 (■) 

;« 3 
2:5 . 1 

31 (•) 
310 

21 4 
112 

:!S 7 
10 1 


8.2 
Xonc 

None 

X'^OIU! 

None 

None 

110 

IS.S 

S.2 

13.0 

5,8 

None 

17 G 

Xone 

None 

r).8 


33 2 


CSHS 


28.2 


CSH 




CSHS 


14 2 


CKR 


20.6 


CRRS 


30.2 


CPR 


20 . 2 


CPRS 


27.4 


CRRN 


31 8 


CRUXS 


20.8 


CSUB 


33 (> 


CSRBS 


IS 6 


CSHB 


31 G 


CSIIIW 


15.4 


CSRL 

CSULS 


14.8 
10.8 


CSHL 

CSIILS 


20. G 









and the oily flavor, ma>- !)>■ (hie to an unl);ilancin,<; of this r(^lationship, the 
0(;currenc(! of these; fiax'ofs beiiio; {\\\c to factoi's in which the presence of 
trimethylaniine in detectable amounts is in no wa>' contributory. 



bacteriolocjical studies 
In an effort to corn^late tlu^ preceding observations with tlu> biological 
aspects of the j^ro'olem, certain bacteriological studies were carried out. 
Thest^ included a bactei'ial analysis of fishy- and non-fishy-fiavored butters, 
and inoculations with pure and mixed cultures into chohne, lecithin, 
buttei'fat, and ci-eam foi- th(> purpose of finding, if possible, an organism 
or a group of organisms whicli in some way n)ight contribute to the develop- 
ment of the fishy flav'or. 



The Lecithin Content of Butter 



137 



Bacterial analysis 

Bacteriological (examinations of fishy- and of normal-flavored butter 
from various sources seemed to show no characteristic differences in flora, 
neither were? the quantitative I'esults consistent. F.ven tho the samples 
examined did not appear to possess marked differences in flora, cultures 
of the predominating type were isolated from the fishy samples for the 
purpose of determining a condition under which they might contribute 
to the characteristic; flavor. The types of l)acteria found included a 
number of acid-iM'oducing varieties, both coccus and rod forms. Among 
the species commonly found were Micrococcus lactis acidi, Mic. lactis al- 
bidus, Bacterium lactis brcins, Bad. aerogenes, and Bact. lactis flocculus. 

The bacteria content of ccu'tain samples of fishy- and of non-fishy- 
flavored butter is given in table 21: 



TABLE 21. Xu.MBEK or Bacteria Found in Various Samples of Fishy- 

Wl) X(iN-l'ISHY-FLAVORKl) BuTTER 



Sample 


Character 

of 

flavor 


Bacteria 

per 

gram 


1 


Strong 

Fishy 

I'ishv 

Fishy 

Oilv 

Fishy 

Fishy 

Oily 

Strong 

TMshy 

Fislu^ 

Strong 

l<ishy 

Storage 

Fishv 

VUhx 


1.200,000 


2 


1. 100,000 


3 


8,000,000 


4 


21, GOO, 000 


5 


30,000,000 


6 


00,000 


7 . . . . 


135,000 


S 


3,000.000 


9 


700,000 


10 


23,500 


11. . . 


8 300 000 


12 


1,000,000 


13 


340 000 


14 


2,000 


lo 


3r)0,ooo 


10. 




405,000 









Inoculationx for the purpose of developing the Jlshij fhvor 

The results of j^n^vious investigations show that littk^ success has 
been attained in the attempt to develop the fishy flavor in butter by 
inoculating the butter itself or by inoculating the cream just prior to 
churning. If the enzymatic idea as already stated in this paper is to be 



138 George Cornell Supplee 

upliold, such ncgativo results can be exi:)lainecl liy the fact that organisms 
inocuhited into the cream just prior to churnino;, oi- into the butter, do 
not have th(> opportunity for growth and consequent ]iroduction of 
bj'-products wliich they would have if allowed to grow in the milk or the 
cream before it is made into butter. There seems to he good evidence 
that there is a i-apid dying-off of tlie bactei-ia in butter after the first 
few days of storage. Furthermore, it is well known that low storage 
temperatures do not entirely prevent enzymatic activity. Rogers (1909) 
shows that, whili^ low iemi)eratures delay the development of the fishy 
flavor, they do not entirely prevent it. In accordance with these facts 
it is conceivable tliat certain enzynu^s winch would be produced by the 
growth of organisms in the cream, and carried into the butter, would there 
continue their activit,y, witli the consequent manifestation of certain 
changes in flavor. Ivcsults obtained in the present investigation indicate 
also the importance^ of a definite acid relationship. With these factors 
in mind, tiie inoculation experiments imdertaken in connection with 
this protilem were carried out in a m:uiner that would alio\v for the 
manifestation of the possibilities indicated. 

Nine organisms, all of which were isolated froin samples of fishy butter 
and Bacteriiun ichlhi/oxun'ux — which Hammer (1917) found would produce 
the fishy flavor in milk — v.ei-e used in these exjx'rinients. The same 
original lot of cream w-.is divided into six parts, and (vich of these parts 
was furtiier divided into ten parts, each of which was inoculated with a 
specific organism. Different methods of handling the six groups of ten 
inoculations each were carried out in such a way that the (>ffect of acid in 
conjunction with the specific organism could b(> determined. Adequate 
cliecks were made from uninoculated cream. Pasteui'ized sweet cream 
was used as tlie basis for all inoculations. When the crcvmi was neutralized 
the acidity was reduced to 0.18 per cent. All samples were made up 
both salted and unsalted, and were scoi'ed aftci' 2:51 days in storage^ at 
a temperature of 0° F. or lovicr. The results of th(» expei'iment as regards 
salted butter are given in table 22. 

It is charactei'istic of the r(\^ults of this experinunit that none of the 
.samples of unsalted l)utt('r showed any of tlie characteristic flavors and 
ai-e therefoi-e not "included in tnble 22. It is believed that the results 
shown in the table clenrly confirm the o|)inion that l)iological agencies, 
particiflarly bacterial enzymes, are responsil)l(i to a great degree for the 
flavors indicated; also, that the fundamental condition necessary for the 



The Lecithin Content of Butter 



139 



TABLE 22. 



Effect on the Flavor of Salted Butte::i, of Inoculating Cream with 
Specific Organisms under Different Conditions 



Sample 


Treatment of cream 


Acidity 
of cream* 


Comments l)y judges 




1( + ) 


2 ( . + ,) 


No. 1 


No. 2 


Xo. 3 


No. 4 


1 


Raw sweet 


0.18 
0.13 
0.3S 

2(; 










2 














3 


Pasteurized sweet, ripened 
Pasteurized swe3t, ripened 

and neutralized 

Pasteurized sweet, acidi- 
fied with lactic acid. . . . 


().3s 










4 










5 


o.rvi 

O.ls 

o.i;] 

0.13 
13 


Fishy 


I'isliy 


Oily 


Fishy 


6 


Pasteurizetl sweet, acidi- 
fied with lactic acid and 
neutralized 


0..-)! 


D BI 


Pasteurized sweet, in- 
oculated with sj)ecifie 
organisms and churned 
at once 

Pasteurized sweet, in- 
oculated with .■^pecific 
organisms and held 21 
hours before churning 

Pasteurized sweet, in- 
oculated with specific 
organisms, held 21 
hours, and neutralizeil 
before churning 










D 100 








Metallic 


D 23 










D 12 


0.13 
0.13 
13 


Tallow^' 
Metallic 






Tallowy 
Oily 


D 18 






D 19 








D 8 


(> K' 








Oily 


D 11 


13 
13 










D 21 










D 10 










E BI 




4.- 










E 100 


41 






Fishv 
Metallic 


Tallowy 


E 23 


0.4.-, 
0.11 
0.41 
0.43 
0.45 
0.43 
0.4(3 
0.43 
0.43 
0.39 


0.41 

0.43 
4.-! 




Fishy 


Oily 


E 12 






E 18 










E 19 


43 










E 8 


4(; 










E 11 


43 




Oily 


Oily 


Fishy 


E 21 


0.43 
39 




E 10 










K BI 


O.IS 
O.IS 
IS 


Metailic 




I'ishy 


Fishy 


K 100 






K 23 








Oily 


K 12 


O.IS 
O.IS 
IS 




Butyric 


Butyric 




K IS 






K 19 










K 8 


IS 










K 11 


0.18 
O.IS 
0.18 




Tallowj- 






K 21 








KIO 






Fishy 





*1 ( -(-) = acidity at time of neutralizinfi 
2 ( -i-) = acidity at time of cliuniing. 



140 



Geokgk Cornell Supplee 

TABLE 22 (,<:oncludcd) 



Sample 


Treatment of cream 


Aciditj- 
of cream* 




Comments by judges 






1( + ) 


2 (+) 


Xo. 1 


Xo. 2 


Xo. 3 


No. 4 


NBI 


Pasteurized sweet, in- 
oculated with si)ecifi(' 
organisms, held 21 
hours, neutralized, and 
ripened 

Pasteurized sweet, in- 
oculated with specific 
organisms, held 2 1 
hours, neutralize! 1, 
ripened, and again neu- 
tralized 

Pasteurized sweet, in- 
oculated with specific 


0.15 
0.41 
0.41 
0.43 
4.- 
0.43 
47 
43 
0.43 
0.39 

0.3(i 
0.2(i 
0.38 
31 
33 
0.32 
. 29 
0.30 
0.32 
0.29 


0.32 

0.3(1 










N 100 










N23 


0.3.-) 
0.37 
0.38 










N 12 










N 18 








N 19 


0..3(> 
33 










N8 










N 11 


29 










N21 


39 










N 10 


0.39 










R BI 

K 100 


O.IS 
1,S 


I'ishy 


Oily 


Fishy 


Fishy 


R 23 


IS 
IS 
IS 








Oily 


R 12 


Butyric 








R IS 








R 1!) 


IS 






Rancid 




R8 


0.18 






Fishv 


R 11 


0.18 








Fishy 


K21 


0.18 










R 10 


IS 

2!t 
4' 










XBI 

X 100 


^h'tallic 


Metallic 


Tallowy 


I'Mshv 

Oily 

Metallic 

Strong 


X23 
X 12 


organisms, held 21 
hours, and ripened 




!() 
3<l 

:;7 

0.3C. 
34 


^h'tallic 
Butyric 


Metallic 
]5utyric 


I'ishy 


X 18 






X 19 










X s 










X 11 


3(1 
.30 
.3(i 










X 21 










X 10 





















*1 ( + ) 1= acidity at time of nf iitralizing. 
2 ("h) ^acidity at time of cliuriiiiiK. 

iii:iiiifestation of (hcso flnvors is grentl.y cnlianct'd by the growth of the 
orgaiiisiiis in the cream; and furthermore, that a definite acid condition 
is essential for tlu> development of tht>S(^ flavors, wliieh aw potentially 
po.ssible from the specific bacteria or en/ymes. In this experiment it 
is ap|)r(;ciated that tht^ results are obtained by an associative action 
with the organisms in tlic starter and those surviving pasteurization; 
this fact, iiowever, do(>s not depreciate the specificity of the jiarticular 
organisms tliat were inoculated. In reviewing the data from this experi- 



The Lecitiiix Content of Butter 



141 



ment, it is interestinc; to note that the sweet pasteurized cream to which 
lactic acid had been added and which was iininoculated, developed the 
fishy flavor. The description of the flavor in Die same sample of butter 
by diffen>nt judges ao;ain calls attention to tlu^ fact that there seem to 
be some conditions common to the fisliy, the metallic, and the oily flavor. 

The most consistent comuKMits fi'om specific organisms seem to be 
from culturc^s BI and 23. Tlu; former- is Bad. ichthyosmius, which was 
obtained from Hammer; tlie latt(M' is an organism isolated from raw- 
ripened-cream butt(u- which develoixnl the fishy flavor after two months 
and retained it for nearly twenty months. 

Butter samples 1'] 23 and X BI were analyzed for trimethylamine 
and ammonia. None; of the former substance was found. Sample X BI 
showed 35.2 parts per million of ammonia, and sample E 23 showed 
17 parts per million. 



loxcxEVIty of bacterium ichthyosmius in butter 
Preliminary expei'iments with Bacterium ichthyosmius indicated that this 
oi'ganism might prcxhice the fishy flavor in butter. It seemed desirable, 
therefore, to determine its longevity in butter made from the inoculated 
cream. Il(>sults of tlu^ (juantitative d(>terminations of the bacteria in salt(>d 
butter containing this organism are sliown in talkie 23; results are given 
only for those sampl(>s to whicli a cliaracteristic flavor was assigned. 

TABLE 23. liACTERiA Content of Salted Butter :\Iade from Cream Inoculated with 
Bacterium ichthyo.smius and Stored at a Temperature of 0° I'\ or, T.nAVKii* 



Ave of sample (davs) 


Xumbor of bacteria pci 


^ .,;...;. 




KBI 


RBI 


X BI 




23,400,000 

11,400,000 

10,4.50,000 

8,500,000 

6,300,000 

1,800.000 

1,1.50,000 

890,000 

750,000 


10,400,000 
10,100,000 
8,300,000 
6,700,000 
6,350,000 
4,000,000 
1,3.10,000 




21 


34,2.50,000 


35 


28,000,000 


49 


17,400,000 


G3 


10,1.50,000 


78. . . 


11,. 500. 000 


91. 


11. 201). 000 


105 


6,400 000 


1!') 




5,400,000 


134 


740.000 

3.55.000 

37.500 




148 






169 




4.600,000 









* These results were furnished liy .J. 1 . i usiok. 



142 



George Cornell Supplee 



Tlu^ decrease in bacteria content of tlie salted butter containing Bad. 
ichlhijosniias shows that, ahho this organism may contribute to the 
dcn'elopment of the fishv' flavor in butter, it does not do so by active 
muUiphcation in that meihum. 

FURTHER Snn)II':rt WITH BACTERIUM ICHTHYOSMIUS 

Tlie resuhs obtained with Baden'uDi /chtlii/osinius seemed to warrant 
a further study of its n^lationship to the fishy flavor in butter. The 
following experiment was carried out wil!i the ])urpose of determining 
the conditions in butter under whi(vh th(^ d(>velopment of the flavor could 
be accelerated. Pasteurized sweet cream was inoculated with this 
organism and held for two days at room temperatui-(\ The butter made 
from this cream was divided into twelve parts, and to each of these parts 
a different substaric(> was added. The cream at the time of churning 
contained 0.23 per cent acid. The treatment of this butter, ;md the time 
of occui'rence of th(> tisliy flavor as determined by two or moi-e judges, 
aiv shown in t;il)l(' 21: 

TABIjE 21. KisHY l*'LAV(Hi AS Dev'Klopku in Butteh W'mrn Was Made from Cream 

IxorUI,ATED WITH IiA("rEHILIM ICHTHYOSMIUS AND TO WhHH V.ARIOUS SUBSTANCES 

Were Added 



Sani])l(' 


Suhstancf added to butttT 


Days in stora'ie 




.52 


94 


136 


17.5 


328 


BI 1 


Nnthinn 




Fishy 


Fishy 


Not s(!ored 


BI 2 


lierkl'eldt filtrate from milk 
culture of Bdd. icbtluiD.'itiiius 

Choliue, 0.0118 per cent 

Calcium caseiuate 








BI 'A 


Oily 










BI 4 










BI.-) 
BI (') 


Lactic acid, 0.117 per cent 
Lecithin from butter 


Fishy 


Fishy 


Fishy 


No 


, scored 
Fi.shv 


BI 7 


Lactic acitl and choliiu; 

Bdct. Inctis (icid> starter 


I'ishy 










BI S 








Slightly fishy 


BI !» 


Berkfeldt filtrate aud caseiuate 
Berkfeldt filtrate and choline. 










BI 10 












BI 11 


Berkfeldt filtrate, lactic acid, 
and choline 












EI 12 


Berkfeldt filtrate made alkaline 




Oily 


Fishy 


No 


J scored 



AH of tlie butters intlicated in table 24 possessed a very disagrei^able 
flaN'or Mild odo!- wIkmi fresh l)ut they seemed to improve in quality during 
storage. The deNclopment of the fishy tiavor in certain samples shows 
some v?]y interesting features. In reviewing the results of this experi- 



The Lecitiiix Context of Butter 



143 



ment it must he homo in mind that tho cream fi'om which they were 
made contained the prochicts of two days jirowth of Bdd. ichthijosmius. 

The development of the fishy flavor in the sami)le to which nothing 
was added, is thxM-efore significant. The earlier occurrence of tlie flavor 
in the sampk^ to which lactic acid was added is significant in that it 
confirms certain observations alr(>ady noted. The development of the 
flavor in the sample containing tho. alkaline Herkfeldt filtrate seems to 
be about sinuiltaneous with its development in the sample to which 
nothing was a<lded. Tlie lack of developm'.nit of the flavor in the samples 
to which the filti'ate was add(>d alone oi- in combination with other sub- 
stances, might api)ear to be contradict oiy to the (^nzyniatic idea pre- 
viously expressed. It, is beli(>ved, however, that this is more than offset 
by the other data, which i)oint to \]\v necessity of a definite set of con- 
ditions that must l)e met in ordei- lo produce the flavor. Such being the 
case, the absence of the flavor when the filtrate was added may be explain(>d 
on the basis that the proper e(iiiilibi-iiun had b(>en disturbed. Th(^ final 
occurrence of fishiness in the sample cotitaining lecithin is of importance 
as indicating that this may be the mother substance of the material causing 
the flavor. Other scattering results do not merit particular discussion 
at this time. 

The trimethylamine and anunonia content of the samples shown in 
table 24, and their acid value, arc; giv(!n in tabk; 25: 

TABLE 25. TuiMirniYhAMrxK .v\*i) Ammonia Content and Acid Value of Butteu 
S.\MPLEs Which Were .\Iai>e from Cream Inoculated with Bacterium ichthyosmujs 
AND to Which Other Substances Were Subsequently Added 



Sample 


Age of 
sjiiiiple 

(daysj 


(CH3)3N 

(parts per 

milliou) 


xib 

(parts per 
niillionj 


Acid 
value 


BI 1 


13G 
328 
328 
328 
94 
328 
328 
328 
328 
328 
328 
136 


7.2 
0.4 
5.8 
4.6 
4.7 
9.4 
8,2 
4 (■) 
") , S 

""us 


20 7 
30 2 
28 4 
26.8 
12 7 
25.4 
24 
22 4 
25^8 

"l7's 


8.7 


BI 2 


9.5 


BI 3 


8 5 


BI 4 


7.8 


BI 5 


8.2 


BI (•) 


8.0 


BI 7 


8.9 


BI S 


8.5 


BI <» 




BI 10 


9.2 


BI 11 . . 


9.4 


p: 12 


9.3 







144 



George Cornell Supplee 



The relatively constant trimethylaniine results shown in table 25 
indicate^ strongly that this substance has been produced in the cream by 
F>a d . ichth yosm ius . 



trimethylamine and am.moxlv prodfctiox by bacterium ichthyosmius 

i.\ milk axd cream 
In order to detorniine tlie triniethyianiine production by Bacterium 
ichthno.smius in milk and cream, oO-cubic-centim(>ter (juantities of these 
substances, sterihztMl, \ver(> inoculated with the organism alone and in 
c()ml)ination with a lactic-acid stai'tca*. Tlu^ inoculations were held for 
foiiy hours at 'M)° ('., and the triin(>thvlaniine and anunonia were then 
determined in 2()-cuhic-ccntimeter ciuantities. TIk^ results of these, 
detei'minations are shown in tal)le 2f): 

TABLE 20. Amotn't ok 'I'ltiMKinYLAMiNE AXD Ammonia Produced in Rkimmilk and 
i\ CuKAM BY Bacterium ichthyosmius 



Inoculalion 



liaclcrluni IchifiiidsrHiit^t and starter 
Bad. ichthi/osmiit.s and starter . . . . 

Bart, irhlhui^sniius 

B(ul. ichlhijosDiius 



Mateiial inocHiiated 



Skiininilk 
Oeam . . , 
Skiminiliv 
C'reaiii . . 



(CHslsN 
(parts per 
milliou) 

Xone 

204.0 

04.4 

74.7 



NH3 

(parts per 
million) 

84 

88 

125 

78 



The results presented in tahle 2(5 are of gr(>at interest as showing beyond 
a doubt that the fishy flnvor pi'oduced in milk and cream by Bdcl. 
iclithno.stniiis is due to t fimethylamine. This being the case, it is obvious 
that this substanc(> would \)v carried into the butter, and ther(% under 
proper conditions which have ah'i'ady been pointecl »out , be r(>sponsil)le 
for the characteristic flavoi- in that material W'itii r(>spect to the pro- 
duction of trimethylamine in cream and in milk by this organism, it is 
desiral)le to again call attention to the ol)S(^ivations of the authoi', in 
which the evolution of a fishy flavor was noted on the addition of alkali 
to sweet cream. 

Thes(> results ai'e of further importance in that the cream inoculatcnl 
with start(>r and Bact. i('hth!!Osmh(s contained a greatei' amount of 
trimethylaniine^ than did tlie cream inoculated with the organism alone. 
This in(Ucatc>s that an aciti condition is most favorable for this particular 



The Lkcithin Content of Buto:r 145 

formentation, whicli would be in hai'mony with tho idea that lecithin 
fvn-nishes the souivf^ of tii(> triniethylamine ])rodii('(Hl by the organism. 
The results are support(Ml also by the fact that lecithin is largely asso- 
ciated with th(> fat, and that according to Hannnarsten and Hcnlin (1915) 
lecithin is decomposed by dilute acids. Such being the case, it is readily 
seen that this fermentation brought about by Bod. ichfhyosmius 
would be greatly enhanced by the presence of acid. Th(> ])resence of 
triniethylamine in skimmilk inoculated with the oi-ganism alone might 
be explained on the basis that the oi-ganism was able to produce this 
substance from [)i'oteins as well as from lecithin. Certain data not 
included in this paper, however, indicate that therc^ is a certain amount 
of lecithin present in skimmilk. Just why there is no ti'imethylamine 
in skimmilk inoculated with Ihe starter and th(> organism, is more difficult 
to explain. It may be that tlu^ gnvitei- acidity in thc> skinnnilk has inhibited 
the particular factor responsible for tiimethylamin(> production. 

PRODUCTION OF THIMETHVLAMIXE FROM LECITIIIN AND CHOLINE BY 

BACTEItlAL ACTION 

In order to determine, if jwssible, whether certain organisms found 
in milk and in butter were capable of decoinjwsing k>cithin or chohne into 
triniethylamine, a series of inoculation experiments were carried out. 
Lecithin alone in 0.3 per cent concentration, and in the presence of 
lactic acid and salt, was inoculated with a number of organisms, some 
of wliich were obtained from milk, souh^ from fishy loutter, and some from 
decomposed egg yolk which had d(>V(>loi)ed th(> fishy Havor. The following 
known species w{M-e also used: Haderiuin lad is ncidi, Bad. oerogenes, 
Bacillus procligiosus, B. ))roteus, Baden'iun ichlfii/osmius, Pscudomonas 
liquejaciens fluoresce ns, Oidium lad/x. All organisms wcw inoculated 
singly and in various combinations, and the cultures were held at 20° C. 
for approximately n'uw months. At the end of that timci the cultures 
were tested for the presence of trimethylamine by berating with alkali. 
Negative results were obtained from all of the lecithin inoculations tested. 
Unfortunately, many of the; culturc^s werc^ contaminated with mold, and, 
since the results could not b(^ considered trustworthy, the}^ were discarded. 

The same series of exfx'riments was repeated by inoculating 0.1-per- 
cent choline alone and in the presence of lactic acid and salt. These 
cultures were held under the same conditions as were the U'cithin inocula- 



146 George Cornell SiprLEE 

tions. Trimoth,ylainin(' \v;is found A\h('i-(> Baderiuin icJilhi/os/nius was 
inoculated alone, in coinbinatiou witli Oidium lacti.'i, and with Bacterium 
ladis acidi. The pi'esence of salt did not seem to ])rev{nit the productiori 
of trimethylaniine. Two organisms wliich were isolated from milk gave 
a pronounced test from the choline inoculation, but gave negative results 
in the presence of lactic acid and sjilt ; Bdcillus produjiosus gave a positive 
reaction from the ch()lin(> alonc^; and Bavierium acrogcnes gave a non- 
typical test under the same conditions, as did Psciidomonas liquefaciens 
fluaresccns. All other iiioculatif)ns gave negative results. 

It would appear fi'om tlie I'esults of th(> inoculation experiments that 
since trimethylaniine is pi'oduced from chohne by Bacterium ichthyosmius 
and cei'tain organisms found in milk, it is (juile possibk^ that the fishy 
flavor and odor found in miUv and in butter may be due to this substance's 
having been ])i-oduced from ilu> clioline of the l(>cithin molecule. The 
fact that the two organisms isolated from milk gave a positive reaction 
and that they were selected at random, indicates that such a fermentation 
might be found fairly oft(>n. These results would tluMx^forc* seem to ))oint 
to Ixicterial agenci(^s as the cause of the fi<!iy fiavoi', its manifestation in 
l)uttei' being dependent on conditions previously mentioned. 

]5A(TEU1AL LXOdLAlloXS L\T() BUTTKRFAT 

A fui'ther attempt was made to pi-oduce the fishy flavor by bacterial 
inoculations into a medium in whicli all the constituents were fairly 
definitcdy known. Pure steiile butterfat from which the i)hosphatides 
had been extracted was used as tiie basis of such a medium. Four series 
of inoculations were made, using, with a f(AV exceptions, the species that 
were inoculated into knathin and choline. These inoculations were stored 
at a tempei-ature of 0° F. or kjwca for two hmuh-cHl and nineteen days, 
at the end of which time th(\v wei'c examincHl for the fishy flavor by four 
judges. Negative^ resuh^ weix- obtained from all inoculations, not one 
of the judges pronouncing any of the 120 samjiles to be fishy in flavor. 
Whether any of tlie samples had jxjssessed the fhvvoi' at sonu; time during 
the storage jxaiod is tliflicult to say. All of the samples had a disagreeable 
tallowy fiavor and odor. The com])osition of each of the four series, 
and the variation in acid value caused 1)v tlu^ inoculations, are shown in 
table 27. All samples contained from 10 to 12 per cent of moisture and 
2 per cent of salt. 



The Lecithin CoiTTEXir of Butter 



147 



TABLE 27. 



Composition of Artifi(;ial Butter and ^'akiations in Acid Value Cai:skd 
BY Bacterial Inoculations 



Sam- 
ple 



1 
2 
3 
4 
5 

6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
If) 
17 
18 
19 
20 
21 
22 
23 
24 
25 
20 
27 
28 
29 
30 



Organism or source 



Bacterium laclis acidi 

Oidium laclis 

Bacterium aerogenes 

Bacterium ichlhyonmius 

Pseudomonas liquefaciens flut\ 

escens 

Bacillus prodigiosus 

Bacillus proteus 

Fishy butter 

Fishy butter 

Fishy butter 

Fishy butter 

Fishy butter 

Egg yolk 

Egg yolk 

Strong buttet 

Strong butt(!r 

Normal butter 

Fishy butter 

Fishy butter 

Fishy butter 

Fishy butter 

Fishy butter 

Fishy butter 

Egg yolk 

Egg yolk 

IVIilk 

:\Iilk 

:Milk 

]Milk 

Check 



Composition and acid value 



Butter- 
fat 



7.8 
7.1 

5.2 

6.8 
8.1 
(i.l 
7.1 
6.1 
6.5 
5.S 



Butterfat 
and 0.15 
per cent 

of lecithin 



Butterfat 

and 

Berkfeldt 

filtrate from 

starter 



8.8 
8.1 
8.1 
6.5 
8.1 
10.4 
9 7 
S.S 
6.8 
7 ,5 
7.8 
6.5 
5.8 



6.1 
5.2 

6.8 
6.8 

7.1 

6.5 

5.8 

7.1 

9.1 

6.5 

6.1 

6.1 

6.5 

6.5 

6.8 

8.1 

6.5 

6.8 

7.8 

6.1 

7.1 

8.1 

6.5 

7.1 

7.1 

6.1 

6.5 

6.8 

8.1 

5.9 



6.5 
7.8 
5.8 
6.1 

7.1 
7.5 
6.5 

7.5 
6.5 
7.8 
7.8 
8 8 
8 4 
8.8 
8 4 
8 4 
8.4 
8.1 
7.8 
8.1 
8.1 
9.4 
8.4 
8.4 
6.5 
7.5 
7.1 
6.1 
8.1 
8.1 



Butterfat, 

Berkfeldt 

filtrate from 

starter, and 

0.15 per cent 

of lecithin 



7.5 
7.8 
7.1 

7.1 

7.1 
6.8 
7.8 
7.5 
7.1 
8.8 
8.1 
8.1 
9.7 
7.8 
8.4 
8.4 
8.8 
9.7 
10.4 
11.0 
7.8 
7.8 
7.1 
7.8 
7.8 
9.1 
10.0 
5.8 
7.4 



The ivsults >ho\vn in table 27 are of interest only to the extent that 
they show the variation in acid valu(> caused by different species of 
bacteria. Inasmuch as the sanipl(>s were placed in storage ininie(liat(>ly 
after being inoculated, it is probahle that the chang(>s are the result of 
bacterial enzynu^s liberated by autolysis, because it has been reix-atedly 
shown that little or no growth takes place during storage. 



148 George Cornell Supplee 

SUMMARY 

The data prcs(>nt(Hl in this pajxT show beyond a doubt that there 
is in jiornial butter a sufficient amount of lecithin to yield, on decom- 
position, small quant it i(>s of trimethylamine, and it is shown also that 
small quantities of this substance are essential for the manifestation 
of a fishy odoi'. Furthermore, it is shown that when this substance is 
worked into butt(^r under the proper conditions, it produces a flavor 
d(\scribed as fisliy. These K^sults are most uniform when trimethylamine 
butyrate is used. An associative fermentation in butter or in cream, 
with the ultimate foi-mation of this substance, is quite possible. As to 
whether or not this or some otlier volatile and unstable combination of 
trimeth,ylamine is tlie cause of th(> natural fisliy flavor, remains to be shown 
more conclusively. Certain data do indicate that trimethylamine is 
found in soni(> samples of fishy-flavored buttei' but not in others. Altho 
it is possible that its presence is incidcMital in such samples, that is not 
believed to be tiie case. In this ccMinection it is worth while to call atten- 
tion to the confusion betw(>en th(^ fishy, oily, and metallic flavors when 
they are pi'escnt to only a slifrht <U^j2;re(\ It seems jwssible that th(> initiation 
of tlie development of these flavors depends on a conunon fundamental 
factor. AMiethei' or not any particular one of tlu>m develops to its typical 
flavor would dcpeMid on tlie presence of ('(M'tain conditions which wei'o 
siHH'ific for that flavoi'. With this possil)ihty in view, it would be logical 
to assume that tiimethylamine is responsibl(> foi- tli(> typical luM'rinjj;, or 
mack(M'el, lla/or and odor in ])utter, and tliat the absence of this su])stance 
would result in the manif(\station of similar but non-typical flavors. 

Tliere sec^ns to l)e no doui)t that the ])res(>nc(> of a definite acid con- 
dition in the ])utter is essential for thc^ (l(>vei()pment of the fishy flavor. 
This condition is best obtained when butter is made from cream con- 
taining lactic acid, regardless of whether this is developed by bacteria 
or added to thi^ cream in the form of the commercial product. Further- 
more, the results indicate that, while a definite acid condition is essential, 
it must be accompanied by some other equally important factor. The 
data show tliat this factor is determined by biological agencies. It appears 
that both th(>se factors nuist (>xist in a definite and delicate relationship, 
and that if the proper equilibi'ium is disturbed, the characteristic flavor 
is not manifest. Numerous results and observations indicate that the 
imknown transient factor is trimethylamine. 



The Lecithin Content of Butter 149 

The bacterioloo;ical aspects of the problem seem to involve the deter- 
mination of the relationship already mentioned. It is shown that the 
acid value of butter is to a certain extent regulated by biological factors, 
probably enzjmies. It is shown also that trimethylamine may be produced 
in milk and in cream, probabl}^ to some extent from lecithin, with the 
consequent production of the fishy flavor in those products. Furthermore, 
it is shown that Bacterium ichthyo.smius, which producc^d the flavor in 
those substances, would produce^ the flavor in ])utt('r also under certain 
conditions. It would therefore schmu possible that other species of 
microorganisms might bring about tlie same type of change. It seems 
highly probable that the growth of bacteria in the cream before it is 
made into butter determines the conditions necessary for the later mani- 
festation of the fishy flavor. 

The data dealing with lecithin as tlu^ source of trimethylamine in milk 
products arc too meager to warrant definit(! conclusions at this time. 
However, the results presented herein, taken together with what is known 
regarding tliis substance, indicate that this is one of the most logical 
sources. 

ackxowledg:\iexts 

The author wishes to express his keen appreciation and gratitude to 
Dr. E. S. Guthrie, Mr. W. E. Ayres, and Mr. H. C. Jackson, of the Depart- 
ment of Dairy Industry at Cornell University, to all other members of 
this Department who have been kind enough to aid in the scoring of 
butter, and to all who have furnished samples for study. 



150 Geokge Cornell Supplee 



BIBLIOGRAPHY 

Barger, George. T1u> simpler natural bases, p. 1-215. (Reference on 
p. 12.) 1914. 

BoRDAS, F., AND 1{a('zkowski, Sig. i)E. Variation de I'acide phos- 
phorique suivant I'age du lait. Acad. Sci. [Paris]. Compt. rend. 
135.302-303. 1902. 

Browne, C. A., Jr. A conti-il)ntion to the chemistry of butter-fat. 
II. The chemical composition of buttcn-fat. Amer. Chem. Soc. Journ. 

21:807-827. 1899. 

Bltdai (Bau(M-). Koloman. Methode zur fiuantitativ(Mi Bestimmuns des 
Annnoniaks und Trimcthvlamins. Ztsclu-. jihysiol. Chem. 86:107- 
121. 1913. 

Davis, W. A. .Vmincs and aunnonium bases. /// Allen's commercial 
or<j;anic analysis 6:1-49. (Ilefen^nce on j). 10.) 1912. 

Fetzer, L. W. The lecilhiii content of milk under jjatliologic conditions. 
Abstracted in Science n. s. 33:339. 1911. 

FoLiN, Otto, and Macallum. A. B. On the determmation of annnonia 
in urine. Journ. biol. chem. 11:523-525. 1912. 

(iLikin, W. Zur bi()l!)i;is('hen Bedeutung des Lecithins. III. Biochem. 
Ztschr. 21 : 348. 1909. 

Hammarsten, Olof, and Hedin, S. O. A text-l)ook of physiological 
clunnisiry. 7th ed. ( Refei'cnce on p. 243.) 1915. 

Hammer, B. W. Msliiness in (>va.oorated milk. lo-.va Agr. Exp. Sta. 
Research bul. 38 : 2;'>3 240. 1917.' 

ILvRDiNfi, H. A., RofiER.s, L. A., and Smith, G. A. Notes on some dairy 
troubl(>s. New York ((i(>neva) Agr. F.xp. Sta. Bul. 183:173-193. 
(Refcn-ence on p. 181.) 1900. 

Harrison, F. C. Rej^ort of Professor of BactfM'iology. Ontario Agr. 
Coll. and Exp. Farm. Ann. rept. 27 (1901) : 74-81. (Reference on p. 79.) 
1902. 

Hasehuoek, Karl. Ueber das Schicksal des Lecithins im Korper, und eine 
Beziehung dessellxMi zinn Sumpfgas im Darmcanal. Ztschr. physiol. 
(4iem. I2:148-l()2. 188S. 

Hunziker, O. F. Investigations in dairy manufactures. In Report of 
the Depaitment of Daii'v Husbandry. Purdue Univ. Agr. Exd. Sta. 
Ann. rept. 28 (1915) :39. " 1910. 



The Lecithin Content ok Butter 151 

Koch, Waldemar. Die Leeithane und ihrc Bcdeutung fiir die lebcnde 
Zelle. Ztschr. physiol. Chem. 37:181-188. 1902-03.' 

Leathes, J. B. The fats, p. 1-138. (Reference on p. 4o.) 1913. 

Long, J. H. Observations on the stability of lecithin. Arner. Chem 
Soc. Journ. 30: 881-895. 1908. 

MacLean, Iluon. ITiitersuchungen iibei' l''ifi-e!b-Lecilhin. Ztsc-hr. phys- 
iol. Chem. 59:223-229. 1909. 

Nerkincj, ,]., AND Haensel, E. Der L(>citliinsehah diM- Milch. Biochem. 
Ztschr. 13:348-353. 1908. 

O'Callaghan, ]\r. A. Fishy-tlavorcHl })utfer. The cause and remedy. 
Agr. gaz. New Soiith Wales 12(1 90 1 ) : ;M 1 -340. 1 902. 

— — ■ — Butter classification. The s(>i<>ntific examination of butt(M- for 
export to l^ngland. Agi-. gaz. Xew South Wales 18 (1907 j : 223-227 
1908. 

Piffard, II. Ci. Fishy flavor in but tea*. Xcnv York pi-od. rev. and Amer. 
creamer}' i3-:20. 1901. 

Reakes, C. ,]., Cuddie, D., axd Riad, II. A. Fishy flavour in butter. 
A preliminaiy note on an incjuirv into the cause of the defect. Journ. 
New Zealand Dept. Agr. 4:1-0. 1912. 

Rogers, L. A. Fishy flavor in butter. U. S. Bur. Anini. Indus. Cir. 
146:1-20. 1909. 

The develoimient of flsliy fla\-or in ])utter. Wisconsin Bu.tter- 

makers Assn. Proc. 13:70. 1911a. 

The fishy flavor in butt (>r. 4'he milk dealer 3"^: 10-12. 1914 b. 



Stanek, Vladi]\iih. Uber das Cholin])erjodid und die quantitative 
Falhing von Cholin durch Kaliumhijoilid. Ztschr. phvsiol. Cluan. 
46:280-285. 1905. 

Washbx'rn, R. M., and DAHLiiERG, A. (■. The influence of salt on the 
changes taking place in storage buttei-. Joui-n. dairv sci. i: 114-12(5. 
1918. . " 



Memoir 24, A Study of the Plant Lice Injurinri the Foliaqc and Fruit of the Apple, the fifth precedinp; 
mimbnr in this soiies of publications, was mailed on October 7, 1910. 

Memoir 25. Tlic Crmn-FHiK of Xeiv York. Purl I . I)istrihi,t,on and TaxoJiomy of the Adult Flies, i\\c 
fourth i)rec('(iinsi number iu thi,* aeries of publications, was mailed on October 23, 1919. 

Memoir 2(), The Dri/ Root-Hot of the Bean, the third preceding number in this series of publications, was 
mailed on October 23, 1919. 



000 885 973 5 



